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Functional characterization of H2-M3-restricted CD8⁺ T cells in innate and adaptive immunity Chow, Michael T. 2010

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FUNCTIONAL CHARACTERIZATION OF H2-M3-RESTRICTED CD8+ T CELLS IN INNATE AND ADAPTIVE IMMUNITY  by Michael T. Chow B.Sc., Simon Fraser University, 2003  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  DOCTOR OF PHILOSOPHY in The Faculty of Graduate Studies (Microbiology and Immunology)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) January 2010  © Michael T. Chow, 2010  ii ABSTRACT The outcome of many host immune responses to perturbations caused by pathogenic infections is dependent on the coordinated actions between innate and adaptive immune cells. Despite the seemingly clear distinction that immune cells belong either to the innate or adaptive immune system, CD8+ T cells that exhibit properties attributed to both compartments of the immune system exist. Specifically, a subset of MHC class Ib molecules, H2-M3, preferentially binds to and presents formylated-peptides to CD8+ T cells resulting in their rapid expansion, quick production of cytokines, and provision of cytolytic protection to bacterial infections. The work in this thesis is focused on the discovery and characterization of the immunoregulatory potential of H2-M3-restricted T cells. I found that the activation of H2-M3-restricted T cells provide rapid enhancement of Ag-specific CD4+ T cell responses following immunization, as well as during the acute, effector, and memory phase following bacterial infection. Immune responses by conventional MHC class Ia-restricted Ag-specific CD8+ T cells were also augmented indirectly by H2-M3-restricted T cells. Using co-culture assays, it was determined that H2-M3-restricted T cells perform these functions by inducing dendritic cell (DC) maturation. Interestingly, H2-M3-restricted T cells were found to be more efficient at performing this function, relative to activated natural killer cells. Therefore, the adjuvantlike properties of innate-like H2-M3-restricted T cells, and possibly other MHC class Ibrestricted T cells, are targets with unique attributes that can be harnessed for future vaccine design strategies to potently focus immune responses against microbial infections.  iii TABLE OF CONTENTS ABSTRACT ..................................................................................................................... ii  TABLE OF CONTENTS .................................................................................................. iii LIST OF ABBREVIATIONS ............................................................................................. x  ACKNOWLEDGEMENTS ............................................................................................. xiv  DEDICATION ................................................................................................................ xvi  CO-AUTHORSHIP STATEMENT ................................................................................ xvii  Chapter 1 Introduction ............................................................................................. 1  1.1   Preface ........................................................................................................ 1   1.2   Classical and non-classical MHC molecules ............................................... 2   1.2.1 Overview of classical MHC molecules ......................................................... 2  1.2.2 Overview of nonclassical MHC molecules ................................................... 4  1.2.3 Human nonclassical MHC class Ib molecules ............................................. 5  1.2.4 Murine nonclassical MHC class Ib molecules .............................................. 6  1.2.5 MHC class I-like molecules........................................................................ 12  1.3   Dendritic cells ............................................................................................ 13   1.3.1 Overview of dendritic cell ........................................................................... 13  1.3.2 Heterogeneity of dendritic cell subsets ...................................................... 14  1.3.3 Immunobiology of dendritic cells ................................................................ 15  1.4   Conventional and nonconventional T cells ................................................ 17   1.4.1 Overview of conventional and nonconventional T cells ............................. 17  1.4.2 Development of conventional T cells ......................................................... 19  1.4.3 Development of unconventional T cells ..................................................... 21  1.4.4 Conventional CD8+ T cells ......................................................................... 23  1.4.5 Conventional CD4+ T cells ......................................................................... 24  1.5   The T cell response to Listeria monocytogenes ........................................ 27   1.5.1 Overview of Listeria monocytogenes ......................................................... 27  1.5.2 MHC class Ia- and Ib-restricted T cell response to LM infection ................ 29   iv 1.6   Thesis rationale, hypothesis, objectives, and significance ......................... 30   1.7   References ................................................................................................ 44   Chapter 2    H2-M3-restricted T cells participate in the priming of antigen-specific  CD4 T cells 1 ....................................................................................... 81  2.1 Introduction ........................................................................................................ 81  2.2 Materials and methods ....................................................................................... 83  2.2.1 Mice and bacteria ...................................................................................... 83  2.2.2 Antibodies and peptides ............................................................................ 83  2.2.3 In vivo mAb depletion ................................................................................ 83  2.2.4 Bone marrow-derived dendritic cells .......................................................... 84  2.2.5 Detection of antigen-specific T cells .......................................................... 84  2.3 Results ............................................................................................................... 85  2.3.1 H2-M3-restricted CD8+T cells participate in the priming of Ag-specific CD4+ T cells ................................................................................................................. 85  2.3.2 CD4+ T cells generated with H2-M3-restricted T cell help improves protective immunity............................................................................................. 86  2.3.3 Effective priming of Ag-specific CD4+ T cells generated with help from nonclassical H2-M3-restricted T cells augment conventional CD8+ T cell responses... .................................................................................................................. 88  2.3.4 Enhancement of memory CD4+ and CD8+ T cell responses by H2-M3restricted T cells ................................................................................................. 90  2.3.5 Qa-1-restricted CD8+ T cells enhance Ag-specific CD4+ T cell responses 91  2.4 Discussion ......................................................................................................... 92  2.5 Acknowledgments .............................................................................................. 96  2.6 References....................................................................................................... 108   v Chapter 3   +  +   H2-M3-restricted CD8 T cells augment CD4 T cell responses by  promoting dendritic cell maturation 1 ................................................. 115  3.1 Introduction ...................................................................................................... 115  3.2 Materials and methods ..................................................................................... 117  3.2.1 Mice ......................................................................................................... 117  3.2.2 Antibodies and peptides .......................................................................... 117  3.2.3 Generation of bone marrow-derived dendritic cells (BMDCs) .................. 118  3.2.4 Generation and purification of H2-M3-restricted CD8+ T cells ................. 118  3.2.5 Generation and purification of IL-2 activated NK cells ............................. 118  3.2.6 In vitro co-culture maturation assay ......................................................... 119  3.2.7 Adoptive transfer experiments ................................................................. 119  3.2.8 Adoptive-transfer experiments ................................................................. 119  3.2.9 Statistical analysis ................................................................................... 120  3.3 Results ............................................................................................................. 120  3.3.1 Simultaneous activation of H2-M3-restricted CD8+ T cells enhances the proliferation of transgenic CD4+ T cells ............................................................ 120  3.3.2 Activated H2-M3-restricted CD8+ T cells induce the maturation of dendritic cells ................................................................................................................ 122  3.3.3 Both soluble factors and cell-to-cell contact contribute to the induction of DC maturation by H2-M3-restricted T cells ....................................................... 124  3.3.4 H2-M3-restricted T cells are more efficient than NK cells at inducing DC maturation ........................................................................................................ 126  3.3.5 DCs co-cultured with activated H2-M3-restricted T cells produced higher levels of pro-inflammatory cytokines ................................................................. 127  3.3.6 DCs matured by activated H2-M3-restricted T cells lower the activation threshold for OT-II T cells ................................................................................. 128  3.4 Discussion ....................................................................................................... 129  3.5 Acknowledgments ............................................................................................ 133  3.6 References....................................................................................................... 144   vi Chapter 4   General discussion and perspectives.............................................. 153   4.1 Adjuvant-like properties of MHC class Ib-restricted T cells .............................. 153  4.1.1 The relationship between conventional antigen-experienced memory CD8+ T cells and nonconventional innate-like CD8+ T cells ....................................... 155  4.1.2 Significance of a heterogeneous memory CD8+CD44high T cell population ... ................................................................................................................ 158  4.2 The in vivo significance of MHC class Ib-restricted T cells .............................. 161  4.2.1 Danger signals mediated by MHC class Ib molecules ............................. 161  4.2.2 Possible human equivalent of H2-M3 molecules ..................................... 164  4.2.3 The autoimmune potential of MHC class Ib-restricted T cells .................. 165  4.3 Concluding remarks ......................................................................................... 165  4.4 References....................................................................................................... 167  Appendix A:   List of publications .......................................................................... 177 Appendix B:     UBC research certificates of approval ....…………………………… 178  vii LIST OF TABLES  Table 1.1  Human and murine MHC class Ia and Ib molecules ................................. 33  Table 1.2  Phenotypic and functional differences between dendritic cell subsets ...... 34  Table 1.3  Phenotypic and functional differences between conventional and innate-like T cells ...................................................................................................... 35  viii LIST OF FIGURES  Figure 1.1  The human and mouse MHC ................................................................... 36  Figure 1.2  Overall scheme of T-cell development in the thymus ............................... 38  Figure 1.3  Model proposing distinct requirements for conventional versus innate-like T cell development in the thymus ............................................................. 40  Figure 1.4  Presentation of Listeria monocytogenes-derived antigens to CD8+ T cells .......................................................................................................... 42  Figure 2.1  Concurrent activation of H2-M3-restricted T cells enhances antigenspecific CD4+ T cell responses ................................................................ 96  Figure 2.2  Improved protective immunity when antigen-specific CD4+ T cells receive H2-M3-restricted T cell help on the same dendritic cell ........................... 98  Figure 2.3  Antigen-specific CD4+ T cells generated with help from non-classical H2M3-restricted T cells augment MHC class Ia-restricted CD8+ T cell responses .............................................................................................. 100  Figure 2.4  Conventional CD8+ T cells cannot enhance LLO-specific CD4+ T cell responses .............................................................................................. 102  ix Figure 2.5  H2-M3-restricted T cells enhance the number of Ag-specific CD4+ and CD8+ memory T cells ............................................................................. 103  Figure 2.6  Qa-1-restricted CD8+ T cells enhance Ag-specific CD4+ T cell responses .............................................................................................. 105  Figure 3.1  Activation of H2-M3-restricted T cells augments DC immunogenicity .... 135  Figure 3.2  Activation of H2-M3-restricted T cells induces dendritic cell maturation .............................................................................................. 137  Figure 3.3  Induction of DC maturation by H2-M3-restricted CD8+ T cells is dependent on cell-cell contact and TNF-α ............................................................... 139  Figure 3.4  H2-M3-restricted T cells are more efficient than NK cells at inducing DC maturation .............................................................................................. 141  Figure 3.5  Increased pro-inflammatory cytokine production by DCs when co-cultured with activated H2-M3-restricted T cells .................................................. 143  Figure 3.6  DCs that have interacted with activated H2-M3-restricted T cells are functionally mature and can enhance CD4 T cell responses, in vitro ..... 144  x LIST OF ABBREVIATIONS α-GalCer  Alpha-galactosyl ceramide  Ag  Antigen  AICD  Activation-induced cell death  APC  Antigen presenting cell  Β2m  Beta-2-microglobulin  B6  C57BL/6  BMDC  Bone marrow-derived dendritic cells  CD122  IL-2Rβ  CD40L  CD40 ligand  CD8EM  CD8 effector memory T cells  CD8CM  CD8 central memory T cells  CFSE  Carboxy-fluorescein diacetate succinimidyl ester  CTL  Cytotoxic t lymphocyte  DC  Dendritic cell  DC-SIGN  DC-specific intercellular adhesion molecule-3 grabbing nonintegrin  xi DN  Double negative  ER  Endoplasmic reticulum  fMIG  fMIGWII  FPR  N-formyl peptide receptor  FPRL1  N-formyl peptide receptor-like 1  FPRL2  N-formyl peptide receptor-like 2  GI  Gastro-intestinal  GM-CSF  Granulocyte macrophage colony-stimulating factor  GMFI  Geometric mean fluorescent intensity  GPI  Glycophosphatidylinositol  HLA  Human leukocyte antigen  HSP  Heat shock protein  ICAM  Intercellular adhesion molecule  IE62  Immediate early 62  IFN  Interferon  Ig  Immunoglobulin  iGb3  Isoglobotrihexosylceramide  xii IL  Interleukin  iNKT cells  Invariant natural killer T cells  ITK  IL-2-inducible T cell kinase  LCVM  Lymphocytic choriomeningitis virus  LFA  Lymphocyte function-associated antigen  LLO  Listeriolysin-O  LM  Listeria monocytogenes  LPS  Lipopolysaccharide  MHC  Major histocompatibility complex  NADH  Nicotinamide adenine dinucleotide  ND1  NADH dehydrogenase subunit 1  NK  Natural killer  NKT  Natural killer T cells  ORF  Open reading frame  PALS  Periarteriolar lymphatic sheaths  PAMPs  Pathogen-associated molecular patterns  PRR  Pattern recognition receptor  xiii pTα  Pre-TCRα chain  Qdm  Qa-1-determinant modifier  RLK  Resting lymphocyte kinase  ROR  Retinoic acid receptor related orphan receptor  SCF  Stem cell factor  SP  Single positive  TAP  Transporter associated with antigen processing  TCR  T cell receptor  TGF  Transforming growth factor  TL  Thymus leukemia  TLR  Toll-like receptor  TNF  Tumor necrosis factor  TRAIL  TNF-related death receptor  Treg  T regulatory  VZV  Varicella-zoster virus  WT  Wild type  xiv ACKNOWLEDGEMENTS I wish to extend my sincere gratitude to my supervisor, Dr. Hung-Sia Teh, for giving me the opportunity to study immunology in his lab and for being incredibly supportive and encouraging. I thank Soo-Jeet Teh for providing me with the immunological tools for conducting research in mice, as well as for being such a caring presence in the lab. Many thanks are given to my thesis Supervisory Committee members, Dr. Marc Horwitz, Dr. Pauline Johnson and Dr. Michael Gold, for their guidance and support. I thank the Canadian Cancer Society and the Canadian Institutes of Health Research for support of my research project through funding provided to Dr. Teh. I would also like to thank the financial support provided by a Canada Graduate Scholarship from the Natural Sciences and Engineering Research Council. I also offer my appreciation to members of my Examining Committee for their time and effort in evaluating my research. My heartfelt gratitude is also directed to fellow scientific colleagues in my lab for their constant support, friendship, and most importantly, great memories. In particular, I thank past and present members of the Teh lab, including Salim Dhanji, John Priatel, Edward Kim, and Xiaoxi Chen for their many insightful scientific discussions and amazing friendship. I would also like to thank my very dear friends in the Microbiology and Immunology department including, but not limited to, Kevin Lin, Kathy Tse, SieLung Tjew, Brian Ruffel, Martin Richer and Helmut Kai for their incredible comradeship, thoughtful discussions, and for positively impacting my Graduate Student experience. Last, but certainly not least, I offer my heartfelt thank you to Dr. Jamie K. Scott, Dr. Mari  xv Montero and Dr. Alfredo Menendez for their years of constant love, encouragement, and incredible support since my years as an undergraduate student at SFU. My deepest sentiments are reserved for my mom and dad, for their years of unforgettable love, moral lessons shared during our lifetime, and calming-silent voice of reassurance. I wish to extend an additional thank you to my mom for being so incredibly strong and instilling in me an immense sense of determination and perseverance when confronted with difficult challenges; I love you. Additionally, I would like to thank my aunt, Joan Lam, and sister, Ryannie Chow for their love and support. The greatest thank you goes to my beautiful wife and best friend, Christine Chow, whose endless love, steady encouragement, unwavering support and unrelenting belief in me, has given me the extra drive to battle through the trying times in both science and life. I am very blessed to have someone so great to share life’s many ups and downs with. I love you with all my heart.  xvi DEDICATION  To my dad, Peter Chow  xvii CO-AUTHORSHIP STATEMENT  Chapter 2 has been published as: Chow, M. T., S. Dhanji, J. Cross, P. Johnson, and H.S. Teh. H2-M3-restricted T cells participate in the priming of antigen-specific CD4+ T cells. J. Immunol. 2006, 177: 5098-5104.  For this paper I contributed all of the experimental design, 80% of the research, all of the data analysis, and wrote the manuscript with editing performed by Dr. Hung-Sia Teh.  The bone marrow-derived dendritic cells were generated by Jennifer Cross. Chapter 3 has been accepted for publication as: Chow, M.T., and Teh, H.S. H2-M3restricted CD8+ T cells augment CD4+ T cell responses by promoting dendritic cell maturation. Eur. J. Immunol. In Press. (DOI: 10.1002/eji.200939934)  For this paper I contributed all of the experimental design, 95% of the research, all of the data analysis, and wrote the manuscript with editing performed by Dr. Hung-Sia Teh.  1 Chapter 1 Introduction  1.1  Preface  This thesis is based on the observation that MHC class Ib-restricted CD8+ T cells exhibit innate-like properties enabling their rapid expansion and production of cytokines that influence the outcome of adaptive immunity. Although CD8+ T cells restricted by H2-M3 molecules, a subset of MHC class Ib molecules, are similar to conventional MHC class Ia-restricted CD8+ T cells since they also utilize the  T cell receptor for antigen recognition, are positively selected in the thymus (1), and provide cytolytic protection to some types of bacterial infections (2), they differ from conventional CD8+ T cells by mounting robust primary but diminished secondary immune responses. This observation suggests that the primary role of MHC class Ib-restricted CD8+ T cells may be confined to the early phase of an immune response. Based on this rationale, it is reasonable to expect that unlike conventional CD8+ T cells, H2-M3-restricted T cells and possibly other MHC class Ib-restricted T cells may have the potential to exert immunoregulatory influences on the developing adaptive immune response. This thesis consists of work that clearly demonstrates the ability of H2-M3-restricted T cells, and an additional T cell subset that is restricted by another MHC class Ib molecule, Qa-1,to augment the immune response of CD4+ T cells. This is a significant observation since CD4+ T cells are arguably at the center of many immune responses because of their multifaceted abilities to support either cell-mediated or humoral immunity. Much of the work described in this thesis demonstrates the ability of H2-M3-restricted T cells to  2 strengthen cell-mediated immunity, and describes the mechanism used by these cells to carry out this unique immunoregulatory function.  1.2  Classical and non-classical MHC molecules  1.2.1 Overview of classical MHC molecules Classical major histocompatibility complex (MHC) molecules are also referred to as MHC class Ia molecules. Table 1.1 shows the common MHC class Ia molecules. In humans, there are three genetic loci that encode for MHC class Ia molecules and individuals who are heterozygous for these three genetic loci can express six versions of MHC class Ia molecules. MHC class Ia molecules are expressed ubiquitously on the cell surface of all nucleated cells at various levels, depending on the cell type. MHC class Ia molecules exists as a heterodimer composed of a heavy chain comprised of three domains (α1, α2, and α3), and an invariant subunit β2-microglobulin (β2m) light chain that is common to all MHC class I molecules (3). In addition to the heavy and light chain, the formation of a stable MHC class I complex on the cell surface requires the binding of a short peptide, normally eight to ten amino acid residues in length, within the peptide binding groove of the  chain. The peptides that bind to MHC class Ia molecules are derived from proteins that are degraded in the cytosol (4). Specifically, the α1 and α2 domains of the heavy chain fold to form two anti-parallel α helices arranged over a platform of anti-parallel β-sheet strands, which together form the  3 peptide binding site, while the α3 domain is associated with β2m and forms a key structure enabling interactions with the T cell accessory molecule CD8+ (4). The genes for MHC class Ia molecules in both humans (HLA-A, -B, and –C) and mice (H-2K, D, and L) are located on chromosome 6 and 17, respectively (Figure 1.1) (5, 6). These genes display a high level of polymorphism due to variations in the amino acid composition of the α1α2 domains and binds not only to endogenous antigens, but also a wide array of foreign peptides enabling the recognition of self and non-self derived proteins (7-9). Most peptides to be loaded on MHC class Ia molecules are generated by degradation of intracellular proteins by the proteasome. In addition, MHC class Ia-binding peptides are also derived from internalized dysfunctional proteins of bacterial or viral origin. The peptides that bind to MHC class Ia molecules are transported to the endoplasmic reticulum (ER) by the transporter associated with antigen processing (TAP) molecule and subsequently loaded onto nascent MHC class Ia molecules under the control of several ER resident chaperons (tapasin, calnexin, calreticulin) (10, 11). The successful assembly of the peptide-MHC complex results in their rapid translocation through the Golgi apparatus to the plasma membrane where they get presented as ligands for recognition by antigen (Ag)-specific receptors of CD8+ T cells, which can then be activated to carry out their effector functions. Due to the extreme polymorphism of MHC class Ia molecules, it is unlikely that all individuals in a population will be equally susceptible to a given pathogen or peptide-based vaccine for immunotherapy.  4 1.2.2 Overview of nonclassical MHC molecules In contrast to the highly polymorphic and ubiquitously expressed MHC class Ia molecules found on the surface of most cells, the MHC locus also encodes several structurally related nonclassical MHC molecules, also referred to as MHC class Ib molecules (Table 1.1) (12). Nonclassical MHC molecules have substantially less polymorphism, a more limited tissue distribution, and a lower level of cell surface expression. Unlike the specialized role of MHC class Ia molecules in presenting peptides from intracellular proteins, MHC class Ib molecules perform a diverse array of immune functions that include the recognition of antigenic lipids, binding and transportation of proteins within the host cell, and the targeting of evolutionary conserved protein epitopes of pathogens (13-15). Additionally, there are even nonclassical gene products whose functions are not related to immunity such as the HLA-H molecule in humans, which is involved in iron metabolism (16). Although to date, the functions of most nonclassical MHC molecules are not fully understood nor appreciated; their evolutionarily preserved presence in both humans and mice is a testament to their importance in vertebrate biological systems. MHC class Ib molecules are encoded by genes linked to the classical MHC genes on chromosome 6 (HLA-E, HLA-F, HLA-G, and HLA-H) in humans and chromosome 17 (H2-M, H2-Q, and H2-T) in mice (Figure 1.1). Additionally, non-MHC class Ia molecules that are encoded by genes outside of the MHC class I locus are referred to as MHC class-I-like molecules (17, 18). Crystallographic data has shown that the structure of MHC class Ib molecules closely resembles that of MHC class Ia molecules, where the extracellular α1α2 structural domain units of nonclassical MHC  5 molecules also form the antigen binding site (19, 20). Despite the structural similarities, there are key differences within the Ag-binding groove that differentially influence their ability to bind different types of peptide ligands. For instance, it has been noted some MHC class Ib molecules bind a diverse array of peptides similar to classical MHC molecules (exemplified by Qa-2), a more narrow set of stressed peptides (exemplified by Qa-1b), formylated peptides (exemplified by H2-M3), or no peptides at all (exemplified by TL, T10, T22) (21-26). The differences in the peptide-binding repertoires between classical and nonclassical MHC molecules may reflect disparate roles for MHC class Ib molecules in immunity. Nevertheless, peptides that bind MHC class Ib molecules can originate from cytoplasmic, nuclear, mitochondrial, viral, or bacterial proteins (27-30). After proteasome degradation, the peptides for some MHC class Ib molecules are shuttled to the ER in a Tap-1 and Tap-2-dependent manner and subsequently displayed on the cell surface (31). 1.2.3 Human nonclassical MHC class Ib molecules The human MHC class Ib family members, HLA-E, HLA-F, and HLA-G typically do not function in conventional peptide presentation (Table 1.1) (32). They are often considered to have a more prominent role in innate immunity and immune regulation (33). Specifically, the best defined MHC class Ib molecules, HLA-E, is highly transcribed in many tissues and can regulate the activation of natural killer (NK) cells by functioning as a ligand for the CD94/NKG2 receptors (34, 35). Instead of binding common peptides derived from intracellular proteins, HLA-E molecules bind only to peptides derived from other HLA class Ia signal sequences as a requirement for cell surface expression (14, 36). The second best studied of the human MHC class Ib molecules, HLA-G, can be  6 expressed as a membrane bound or soluble form and has long been postulated to be a mediator of maternal-fetal tolerance because of its predominant expression on not only trophoblast cells, but also on virtually all cellular interfaces where maternal and fetal cells come into contact (37-39). HLA-G has been shown to inhibit NK cell activity likely through interactions with immunoglobulin (Ig)-like transcript (ILT) ILT2 and ILT4 receptors (40, 41). The least well studied of the three human MHC class Ib molecules, HLA-F, interacts with ILT2 and ILT4 receptors and is postulated to have a minor role in mediating maternal-fetal tolerance (42). This was confirmed when HLA-F, normally expressed in the cytoplasm, was detected on the cell surface with the progression of pregnancy into the second trimester (43). 1.2.4 Murine nonclassical MHC class Ib molecules Unlike classical MHC molecules, certain nonclassical MHC molecule subsets are expressed as a membrane bound or secreted form, contain shorter cytoplasmic tails, or lack consensus residues associated with peptide binding (44, 45). These oligomorphic molecules have been described in many organisms, but are best characterized in mice. The murine genome contains greater than 40 class I genes, the majority of which are encoded within the nonclassical H2-Q, H2-T and H2-M regions, which contains approximately 10, 20, and 18 genes, respectively (Figure 1.1) (8, 44, 46). Approximately 40-50% of these genes encoding mouse MHC class Ib molecules have an open reading frame (ORF) and are expressed at the transcriptional level, while the remainder are unexpressed pseudogenes (47-49). Table 1.1 illustrates the types of MHC class Ib molecules present in mice. Although nonclassical MHC molecules are involved in  7 immunological functions like classical MHC molecules, they generally serve specialized roles during immune responses. The structurally best defined MHC class Ib molecule encoded within the H-2Q region, Qa-2, is expressed in a wide variety of tissues and can either be bound to the cell membrane by a glycophosphatidylinositol (GPI) anchor or secreted by activated T cells, both of which requires TAP and β2m (50, 51). Although the functions for Qa-2 molecules have not yet been elucidated, they have been shown to bind certain peptides with very stringent binding specificities, relative to classical MHC molecules. Furthermore, it has been demonstrated that the expression of Qa-2 results in MHCunrestricted killing of target cells (29). Interestingly, in contrast to MHC class Ia molecules, the α3 domain of Qa-2 cannot bind to CD8 molecules, which alludes to the possibility some Qa-2-specific CTLs may recognize and become activated by Qa-2 directly by virtue of having a high affinity TCR. As this may present an autoimmune threat, switching from the membrane-linked to the secreted form has been theorized to make Qa-2-expressing host cells refractory to killing by Qa-2-specific CTLs (8). In addition to immunological functions, other studies suggested a role for Qa-2 during early development by influencing the rate of embryonic implantation and subsequent survival (52-54). In addition to Qa-2, the H2-Q region also encodes the MHC class Ib molecules Q9 and Q10. Q9 antigens are expressed in most tissues, including immunologically privileged sites such as the eye (50). These Q9 molecules have also been shown to be involved in anti-tumour responses by inducing CTL responses in syngeneic host (55). Unlike Q9 molecules, the function of Q10 has remained more enigmatic. However, recent studies determined that Q10 molecules express peptide-binding sites that  8 resemble those expressed by classical MHC molecules H-2K, D, and L, alluding to a possible role in antigen presentation to T cells (56). Of the approximately 20 genes assigned to the mouse H2-T region, reports have identified 11 MHC class Ib genes that are transcribed in the C57BL/6 (B6) mouse, some of which contain greater than 85% sequence similarity in the coding region indicating possible function redundancy (8, 57). The thymus leukemia (TL) antigen, also termed H2-T3, is encoded by T3 and T18 and was the first MHC class Ib molecule to be discovered (42). The initial reports indicate that TL molecules were expressed only on normal thymocytes or thymic leukemia cells. However, more recent reports have also identified the presence of TL antigens in the small intestines (58). TL antigens can prime γδ T cells resulting in their release of granules containing serine esterases, which suggests a role in mucosal immunity (96). The expression of TL is β2m-dependent, TAP-independent, and TL-specific αβ or γδ CTLs can recognize the α1α2 domain unit in a peptide-independent manner (24, 58, 59). In addition to TL antigens, the H-2T region also encodes the gene products T22 and T10, which are closely related and have been shown to function as ligands for γδ T cells (8). T22 is expressed constitutively in a variety of different cell types, while the expression of T10 is inducible in cells of the immune system. All strains of mice express T10. However, functional T22 molecules is absence in BALB/c and C3H mice (60). Interestingly, they are both associated with β2m but do not appear to bind peptide ligands, even though these molecules are recognized by γδ CTLs (26, 60). Perhaps the best-known MHC class Ib molecule encoded in the H-2T region is Qa-1 that is encoded by the gene T23, which is orthologous to human HLA-E (61).  9 Among the 10 alleles identified for Qa-1, the best-defined molecule is Qa-1b. Unlike HLA-E, whose expression appears to be dependent on TAP function and is stabilized by binding appropriate leader sequences that serve as peptides, Qa-1b can be expressed in the absence of TAP. Furthermore, Qa-1b is not stabilized by the addition of peptides derived from the leader sequence of H2-D or L molecules, termed Qa-1 determinant modifier (Qdm) (35, 36, 62). Similar to HLA-E molecules, Qa-1b molecules are widely expressed in many mouse tissues, associate with β2m, and can present peptide Ags from self or foreign proteins to elicit strong CTL responses (63, 64). Although Qa-1b is expressed on a variety of hematopoietic tissues, it is expressed at very low levels on resting T cells and thymocytes, which can be significantly upregulated following peripheral T cell activated by antigen (65). To delineate the significance of this, mice deficient in Qa-1b were studied and found to suffer from exaggerated secondary CD4+ responses after viral infection or immunization with foreign or self peptides, suggesting a role for Qa-1b in guarding against the expansion of pathogenic autoreactive CD4+ T cell populations and consequent autoimmune disease (66, 67). Qa-1b-specific CD8+ T cells are thought to mediate suppression by interacting with Qa-1b molecules on either antigen presenting cells (APC) or on the activated CD4+ T cell or B cell surface (68). There are also descriptions of Qa-1b-restricted CD8+ T cells capable of killing activated Vβ8+ T cells to mediate immune regulation (69). In addition to immune regulation, Qa-1b has been shown to act as ligands for CD94/NKG2 heterodimers, receptors that are expressed by NK cells, NKT cells, and a small fraction of αβ and γδ T cells. This particular receptor-ligand interaction has been shown to regulate cytotoxic killing of infected, stressed, or transformed cells (35, 70-73).  10 NK cell-mediated lysis can proceed or be inhibited depending on whether NKG2A or NKG2C, complexed with CD94, is interacting with the Qa-1b/Qdm complex (35, 71). It has also been suggested that the presence of NK receptors on some αβ and γδ T cells may be to fine-tune the T cell response by raising or lowering the threshold of TCR triggering (74). Qa-1b has also been demonstrated to bind to heat shock protein (HSP) HSP65 peptides after the tryptic digestion of Mycobacterium bovis, suggesting that Qa1b molecules may have evolved to bind HSP-derived peptides (75). More recently, CD8+ CTLs with specificity for the bacterial epitope GroEL, which cross-reacts with murine HSP60 secreted by stressed macrophages, were generated in mice after infection with Salmonella typhimurium(75, 76). This observation indicates the involvement of Qa-1b in bacterial infections, which is supported by the observation that Qa-1b-restricted CTLs are detected in mice that have been infected by the intracellular bacterial pathogen Listeria monocytogenes (LM) (77). Of the approximately 18 genes in the H2-M region, little is known about the regulation and possible function of the majority of them with the exception of M3 (23). The M3 molecule, also termed H2-M3, was discovered as a minor histocompatibility Ag, which associates with β2m and functions as a MHC-restricted element for CD8+ T cells. They have a 10,000-fold selectivity for N-formyl peptides, relative to non-formylated peptides. Based on this observation, combined with the fact translation in prokaryotes is initiated with N-formyl-methionine rather than amino-methionine, the selectivity of H2M3 was predicted and confirmed to be specialized for presenting bacterial peptides to αβ T cells (1, 78, 79). However, the requirement for a formylated N-terminus is not absolute, but rather a matter of affinity (80). This would explain the ability for H2-M3  11 molecules to present nonformylated viral epitopes to CTLs, albeit with less efficiency compared to formylated peptides (81). H2-M3 molecules are also able to bind endogenous sources of natural ligands derived from the N-terminus of NADH dehydrogenase subunit 1 (ND1), which is mitochondrially encoded (27, 82, 83). Due to the lack of endogenous formylated methionine-containing peptides that could arise only from mitochondrial proteins in the steady state, with the exception of B cells, the surface expression of H2-M3 is undetectable in most cell types (84, 85). Despite the lack of cell surface expression, H2-M3 molecules are held in the ER in a peptide receptive state and can therefore traffic rapidly to the cell surface either by the addition of exogenous peptide or presumably infection by intracellular bacteria, in mainly a TAP-dependent manner (86, 87). However, reports have also shown that, given a supply of peptides from phagosomes after infection by LM, H2-M3 can be properly folded and exported to the cell surface in a TAP-independent manner, which suggests different requirements for endogenous and exogenous N-formylated peptide sources (23). To date, there are three LM Ags presented by H2-M3 molecules to their respective CTLs: fMIGWII is derived from the LemA transmembrane protein, fMIVIL is derived from an unknown protein source, and fMIVTLF is derived from the AttM transcriptional attenuator polypeptide (88-90). Since most of the peptide-binding specificity of H2-M3 is associated with the N-formyl terminus, H2-M3-restricted CD8+ T cells exhibit crossreactivity with different bacterially derived N-formylmethionine peptides (91). Thus, in addition to LM infection, H2-M3-restricted T cells are also found to be expanded after infection with Mycobacterium tuberculosis (92).  12 1.2.5 MHC class I-like molecules Several other MHC class I-related genes, termed MHC class I-like molecules, have been discovered. However, unlike MHC class Ia and class Ib molecules, they reside on chromosomes other than those carrying the MHC genes (93). The best-studied MHC class I-like molecule, CD1, can be found in both humans and mice. CD1 molecules have a similar structure to MHC class Ia molecules, but rather than presenting peptides, they bind lipids and glycolipids and present them to their respective T cells (94). Based on sequence homology, there are five known isoforms of CD1 molecules classified into one of two categories. Group I CD1 molecules consists of CD1a, CD1b, CD1c, and CD1e molecules found in humans, and group II CD1 molecules consists of CD1d molecules found in both humans and mice (95). Group I CD1 molecules are primarily expressed by cortical thymocytes and to a limited extent extrathymically on cells of the hematopoietic lineage (13, 96-100). Each member of the group I CD1 molecules were found to present lipids or glycolipids, and were found to have distinct trafficking patterns within the cells that express them (101). In contrast to group I CD1 molecules, group II CD1 molecules are expressed weakly in the thymus and is found expressed primarily extrathymically by a wide number of tissues, which suggests a likely role in immune function (102). Confirming this function, CD1d has been demonstrated to present both endogenous and bacterial glycolipids to NKT cells, which express both NK cell receptors and a restricted set of αβ TCRs, leading to their activation and contribution towards antimicrobial immunity (103-105). Under physiological conditions, NKT cells restricted by CD1d molecules that express the invariant TCR α-chain, encoded by Vα24-JαQ genes in humans and Vα14Jα28 in mice (iNKT cells), can recognize lipids  13 derived from bacterial sources in the context of CD1d to provide anti-bacterial immunity (103, 105). More recently, the endogenous ligand that NKT cells recognize was identified to be isoglobotrihexosylceramide (iGb3), which is a lysosomal glycosphingolipid (106). Interestingly, under less physiological conditions, α-galactosyl ceramide (αGalCer), which is a lipid purified from sea plants not found in either humans or mice, has been discovered to be a very potent agonist capable of activating most NKT cells when presented by CD1d molecules. Furthermore, αGalCer has been shown to interact with dendritic cells (DC), which are central regulators of adaptive immune responses (105, 107-109).  1.3  Dendritic cells  1.3.1 Overview of dendritic cell DCs are an integral part of the immune system that functions as a bridge that link innate immune responses with the development of adaptive immunity. They are widely distributed and exist in four developmental stages that consist of bone marrow progenitors, precursor DCs that traffic through the blood and lymphatics, tissue-resident immature DCs that possess high endocytic and phagocytic potential, and as mature DCs that are present within secondary lymphoid organs (110, 111). The acquisition of T cell stimulatory potential by DCs is dependent on the upregulation of class I and II MHC molecules, costimulatory as well as adhesive molecules, which can be achieved by responding to stimuli from their milieu, as well as by interacting with other innate  14 immune cells (112-114). In addition to the induction and control of T-cell immunity, they are also involved in the modulation of B cell and natural killer (NK) cell responses. Furthermore, under different cues present in the microenvironment, DCs can induce not only immunity, but also Ag-specific unresponsiveness or tolerance in central lymphoid organs as well as in the periphery (115, 116) 1.3.2 Heterogeneity of dendritic cell subsets Conventional DCs develop in vivo from hematopoietic precursor cells (reviewed in (110, 117)) whose lineage derivation can generally be divided into the lymphoid, myeloid and plasmacytoid subsets, the latter of which are considered unconventional DCs and are mainly involved in antiviral immunity (118-124). Table 1.2 illustrates the phenotypic and functional differences between the different DC subsets. Lymphoid DCs are found in the T-cell rich areas of the periarteriolar lymphatic sheaths (PALS) in the spleen and lymphoid organs (125). By contrast, myeloid DCs are found in the sub-epithelial dome of the Peyer’s patch, the subcapsular sinus of lymph nodes, and the marginal zone of the spleen, but can be induced to migrate to the PALS under the influence of proinflammatory signals (118, 126). Both subsets express high levels of CD11c, MHC class II, and the costimulatory molecules CD86 and CD40. They can be distinguished from each other by CD8α, which is expressed as a homodimer on the lymphoid DCs and not on myeloid DCs (127, 128). The lymphoid DCs produce higher levels of IL-12, a key cytokine for the development of cell-mediated adaptive immunity, and are less phagocytic relative to myeloid DCs (129, 130). Additionally, lymphoid DCs were reported to prime allogeneic CD4+ and CD8+ T cells less efficiently than myeloid DCs (131, 132). However, both lymphoid and myeloid DCs appear to prime Ag-specific CD4+  15 T cells efficiently, in vivo (133, 134). Although the absolute relationship between lymphoid and myeloid DCs remains unclear, much of what is known about the biology of DCs has been acquired through the in vitro generation of DCs from precursor cells. The hematopoietic growth factor, Flt3 ligand, has been shown to generate DCs from CD34+ bone-marrow progenitor cells, with phenotypic characteristics similar to both lymphoid and myeloid DCs (135, 136). In contrast, the generation of murine lymphoid DCs requires the treatment of thymic lymphoid precursors with a combination of IL-1β, TNFα, IL-7, stem cell factor (SCF), and IL-3 cytokines, whereas murine myeloid DCs can be generated by treating bone marrow precursors or peripheral blood monocytes with granulocyte macrophage colony-stimulating factor (GM-CSF), or GM-CSF plus IL-4 (137-140). 1.3.3 Immunobiology of dendritic cells In the absence of external stimuli, DCs exists as immature cells primarily involved with Ag capture, have low to moderate expression of MHC class II and costimulatory molecules (CD40, CD80 and CD86), and are believed to be capable of inducing tolerance. Immature DCs carry out their endocytic roles by constantly sampling the surrounding environment for pathogens including, bacteria, viruses, fungi, protozoan and metazoan parasites (141). The recognition of these evolutionary conserved pathogen-associated molecular patterns (PAMPs) by DCs takes place through the use of toll-like receptors (TLRs), which are encoded by the germline. For instance, the recognition of the major cell wall component of Gram-negative bacteria, lipopolysaccharide (LPS), involves TLR4, while the recognition of viral double-stranded RNA involves TLR3. Engagement of TLRs on DCs results in several pleiotropic effects  16 on DCs including augmented DC survival, chemokine secretion, expression of chemokine receptors, migration, cytoskeletal and shape changes associated with endocytic remodeling, which enable them to promote T cell activation, clonal expansion, and differentiation (142). Interestingly, many signals have been shown to induce some aspects of DC maturation in addition to TLR signaling, which involve the exogenous addition of inflammatory cytokines, such as interferon-α (IFN-α), IFN-β, tumor necrosis factor α (TNF-α) and IL-1 (143-147). But unlike DCs that undergo maturation as a result of PAMP/TLR interactions, which can prime an effective CD4 T helper type 1 (TH1) response, inflammatory cytokines can only help DCs to amplify but not initiate adaptive immune responses (148). DCs migrate into T cell areas of lymphoid organs following Ag exposure and activation, a process regulated by the upregulation of CXCR4 and CCR7, which promotes their migration through the bloodstream into lymphoid organs enabling interactions between DCs and T cells (149-151). The cell surface of DCs also express receptors such as DC-SIGN (DC-specific intercellular adhesion molecule (ICAM)-3 grabbing non-integrin), which is crucial for mediating physical contacts between DCs and T cells enabling DCs to screen numerous T cells for an appropriately matched TCR (152). In addition to DC-SIGN, adhesion receptors such as lymphocyte-functionassociated antigen (LFA)-1, ICAM-1, LFA-3 and CD44 may also be expressed on mature DCs and promote adhesion to T cells (153). In addition to adhesion molecules, the soluble cytokines secreted by DCs, which varies with the nature of the stimulus, maturation stage of DCs, interactions with other activated effector cells, and the existing cytokine microenvironment, have a significant impact on influencing the outcome of  17 adaptive immune responses. For instance, antigens that prime DCs to secrete IL-12 will typically induce conventional T cell differentiation into the TH1 lineage (cell-mediate immunity), whereas antigens that do not elicit, or inhibit IL-12 production, will promote development towards the TH2 lineage (humoral immunity) (113, 154-157).  1.4  Conventional and nonconventional T cells  1.4.1 Overview of conventional and nonconventional T cells Conventional T lymphocytes, also referred to as classical MHC-restricted T cells, constitute cells of the adaptive immune system and are central mediators of cellmediated immunity. They recognize specifically fragmented linear antigenic peptides as a complex with MHC molecules, via their TCR. The two major subsets of conventional T lymphocytes, recognizing antigenic peptides presented by MHC class Ia- and class IIrestricted MHC molecules, are cytotoxic CD8+ T cells and helper CD4+ T cells, respectively. These two T lymphocytes each have distinct roles in adaptive immune responses. Whereas conventional CD8+ T cells are capable of protecting the host by inducing the death of transformed tumor cells or cells infected with bacteria or viruses, conventional helper CD4+ T (CD4 TH) cells perform a number of other key roles, some of which includes establishing, maximizing, and maintaining the proper functioning of the immune system. CD4 TH cells can be further divided into TH1, TH2, TH17 and T regulatory (TReg) cells based on the types of cytokines they produce. TH1 cells primarily produce interferon gamma (IFN-γ), participate in cell-mediated immunity, and are  18 essential for controlling intracellular pathogenic infections. TH2 cells primarily produce IL-4 and provide help for B cells, which are essential for antibody-mediated immune responses for controlling extracellular pathogenic infections. TH17 cells primarily produce IL-17 and IL-22, and are found at the interface between the external and internal environment, such as the skin and gastro-intestinal (GI) tract, where they mediate the clearance of extracellular bacteria from those surfaces. TH17 cells have also been implicated as potent effectors cells contributing to several autoimmune disorders. In contrast to the ability of TH1, TH2 and TH17 cells to promote the clearance of pathogens as their main effector functions, TReg cells produce the immunosuppressive IL-10 and transforming growth factor β (TGF-β) cytokines and through additional mechanisms can suppress and/or regulate the extent of adaptive immune responses, thereby contributing to the maintenance of immune system homeostasis and tolerance to self-Ags. In contrast to the well-studied conventional T cells, nonconventional or nonclassical MHC-restricted T cells, are less well studied but have been demonstrated to possess both innate and adaptive immune characteristics. Table 1.3 lists the differences between classical and nonclassical CD8+ T cells. They express a restricted set of semi-invariant, gene rearranged TCR receptors with limited antigen diversity, suggesting that they recognize conserved structures, rather than a wide array of diverse Ags (158). Nonconventional T cells exhibit a natural memory phenotype, which may be what enables them to respond by producing cytokines in the presence of tissue perturbations with innate-like characteristics. Interestingly, in addition to their ability to recognize conserved non-self Ag of microbial origin, they can also recognize a small set  19 of conserved stress-induced self-Ags (159). Furthermore, a subpopulation of nonconventional CD8+ T cells expressing high levels of IL-2Rβ (CD122) has been identified in naïve mice to have the capacity to directly regulate immune responses, mediated by both conventional CD8+ and CD4+ T cells, in an IL-10-dependent manner (160, 161). However, there is some controversy regarding this observation, which needs to be confirmed by other laboratories. Although the exact function of nonconventional CD8+ T cells has remained enigmatic, it is becoming clear these cells have important regulatory roles in the context of various immune responses, including the maintenance of tolerance, inhibition of tumor development, protection from autoimmune diseases, as well as the augmentation of immune responses against bacterial and viral infection (159, 162-166). To aid in their study, mice genetically deficient for all MHC class Ia genes but which express β2m and class Ib molecules have been made. These mice lack conventional CD8+ T cells, which are dependent on MHC class Ia molecules for their development, but possess nonclassical MHC-restricted T cells and therefore serve as an excellent model for deciphering the function of these cells (167). 1.4.2 Development of conventional T cells T cell development takes place in the thymus, an organ that provides the necessary microenvironment enabling the production of T lineage committed thymocytes from bone marrow (BM)-derived progenitor cells, in three distinct phases consisting of lymphopoiesis, TCR-mediated selection, and functional maturation (Figure 1.2) (168). The distinct stages of T cell development can be identified by the expression of the cell surface molecules CD3, CD4, CD8, and CD25. The earliest precursor cells that arrive in the thymus, lacking most of the cell surface molecules that are readily detectable on  20 mature T cells, physically interact with thymic stromal cells. These CD4-CD8- double negative (DN) progenitors undergo a stepwise process characterized by the acquisition and loss of the cell surface molecules CD44 and CD25. The cells progress from the DN1 (CD44+CD25-) stage to the DN2 (CD44+CD25+) stage and then to the DN3 (CD44CD25+) stage, which are all accompanied by vigorous cell proliferation and stepwise movement of the cells from the cortico-medullary junction of the thymus towards the cortex and subcapsular regions (169, 170). Once the progenitor cells enter the DN3 stage, they lose the ability to differentiate into other immune cells such as B and NK cells, and are absolutely committed to the T cell lineage (171). It is also at this stage where thymocytes initiate TCRβ gene rearrangement, and upon successful pairing with the invariant pre-TCRα chain (pTα), these developing thymocytes can progress into the DN4 (CD44-CD25-) stage (171). The generation of a functional TCRβ/pTα complex results extensive proliferation accompanied by the expression of both CD4+ and CD8+ coreceptors, and rearrangement of the TCRα locus such that the final rearranged TCRs will be selected on the basis of having some reactivity to self-peptide presented in the context of self-MHC molecules (172). Any T cells expressing receptors that cannot bind to self-peptide/MHC will not receive any survival signals and will die by neglect. At the other end of the spectrum, any T cells expressing receptors that bind too strongly with self-peptide/MHC will be eliminated by negative selection to protect the host from autoreactive T cell clones. Only T cells that undergo positive selection to ensure that they can recognize foreign peptides in the context of self-MHC molecules, while maintaining tolerance to self-Ags, will survive past this stage, mature, and exit the thymus as naïve CD4 or CD8 single positive (SP) T cells (172).  21 The strength of TCR signals transmitted to a given thymocyte dictates survival and lineage choice. Several signaling molecules proximal to the TCR have been implicated in the modulation of TCR signaling for the maturation of conventional CD4+ and CD8+ T cells. Among them are the MAP kinases, particularly ERK, which play a key role during positive selection, and JNK and p38, which are involved in negative selection (173-175). In addition to these signaling molecules, the strength of signaling through the tyrosine kinase p56lck is involved in the commitment of developing thymocytes into the CD4+ (strong p56lck activation) or CD8+ lineage (weak or no p56lck activation) (173). Recent studies have also identified several molecules important for CD4+ lineage development, which include Lck (176), GATA-3 (177), RasGRP1 (178) and Th-Pok (179, 180)(179, 181). By contrast, members of the TEC kinase family, ITK (IL-2-inducible T cell kinase) and RLK (resting lymphocyte kinase) (182) and RasGRP1 (183) are required for the development of conventional naïve CD8+ T cells. (182). 1.4.3 Development of unconventional T cells Similar to the development of conventional T cells, the development of unconventional T cells is also regulated by multiple independent signals derived from interactions between thymocytes and thymic stromal cells, which include the antigen specificity of the TCR, as well as the strength of signaling through this receptor (159, 184, 185). Recent collections of work spanning divergent areas of research provides evidence that DP thymocytes give rise to many lineages of mature T cells in addition to conventional CD4+ and CD8+ T cells. The two best-studied examples of alternative, nonconventional lineages are those that lead to the development of Treg cells and CD1d-restricted NKT cells (186-188). In addition to these two cell types, other non-conventional T cells  22 including CD8+ T cells with TCRs specific for nonclassical MHC class Ib molecules such as H2-M3 and Qa-1b, are believed to be derived from DP progenitors (1, 189, 190). Unlike the selection of MHC class Ia-restricted T cells by non-hematopoietic thymic epithelial cells, both hematopoietic cells and non-hematopoietic cells can select for non-conventional MHC class Ib-restricted T cells(Figure 1.3)(1, 190, 191). These differences in thymic education, likely as a result of relatively high affinity interactions between the TCR and the selecting ligands, has been proposed to contribute to the observed activated-memory phenotype expressed by these nonclassical MHC-restricted T cells (192). Associated with this activated phenotype is the ability of these cells to respond with “innate-like” kinetics to perturbations in tissue homeostasis (32). Furthermore, the constitutive expression of transcription factors that regulate effector genes also enable nonconventional T cells to exhibit immediate effector functions such as cytokine production (182, 193-196). Since this subset of mature T cells can acquire effector functions as a result of their maturation process, rather than as a consequence of their activation, proliferation, and differentiation events that occur subsequent to antigen encounter in the periphery, these nonconventional T cells are referred to as “innate-like” T cells (182). Based on these characteristics, CD1-, H2-M3- and Qa-1brestricted T cells are considered to be innate-like T cells. Interestingly, unlike the dependence on the TEC kinases ITK and RLK for the development of conventional T cells, nonconventional T cell development is crucially dependent on both IL-15 cytokine and the IL-15 receptor (IL-15Rα) (182, 197-199). Although H2-M3- and Qa-1b-restricted T cells have not been examined for IL-15 dependence, they are hypothesized to be dependent on IL-15 based on overall similarities between these cells and the well-  23 studied CD1-specific NKT cells and the absence of all nonconventional T cells in IL-15deficient mice (182, 199). 1.4.4 Conventional CD8+ T cells As a result of thymic selection, naïve T cells express a TCR repertoire that enables them to respond to most pathogens. The activation of naïve MHC class Ia-restricted CD8+ T cells is controlled by DCs that provide at least two signals for their activation. Signal 1 is provided by strong TCR interactions to pathogen peptides presented by selfMHC molecules, whereas signal 2 is delivered via T cell costimulatory receptors that interact with complementary costimulatory ligands on APCs (200, 201). Upon receipt of these two sets of signals in the context of an immune response, naïve CD8+ T cells proliferate, synthesize cytokines, and differentiate into effector cells that leave secondary lymphoid organs to travel to the site of infection to carry out their antimicrobial functions, which include killing of infected cells. Amazingly, one precursor CD8+ T cell can give rise to more than 10,000 daughter cells over the course of 5 – 8 days (202, 203). The expanded T cells then undergo a contraction phase that eliminates 90-95% of pathogen-specific effector T cells with the few surviving making up the memory CD8+ T cell population, which provides the host with long-term immunological protection against future challenges with the same pathogen (202-204). Two populations of memory CD8+ T cells have been described according to their recirculation pattern: central memory T cells and effector memory T cells, which preferentially reside in secondary lymphoid organs and peripheral tissues, respectively (205, 206). In general, memory CD8+ T cells are able to rapidly secrete IFN-γ upon restimulation, but do not contain sufficiently high levels of Granzyme B for them to retain  24 their cytolytic function, which suggests that they may serve a complementary function by support innate immune cells responses(207). Furthermore, memory CD8+ T cells require lower concentrations of Ags and less costimulatory signals relative to naïve CD8+ T cells for their activation (208). The maintenance of these Ag-experienced memory CD8+ T cells is a dynamic process, which involves homeostatic turnover, characterized by IL-7- and IL-15-driven proliferation balanced by cell death (209-211). These pathogen experienced conventional memory CD8+ T cells are distinct from nonconventional CD8+ T cells, since the latter is believed to acquire their memory phenotype as a consequence of their development (32, 197). Accumulating evidence support the absolute requirement for CD4+ T cells in the generation of fully functional CD8+ T cells. Memory CD8+ T cells that develop in the absence of CD4+ T cells are able to mediate effector functions upon restimulation. However, they express high levels of TNF-related death receptor (TRAIL), failed to undergo a second round of clonal expansion, and undergo activation-induced cell death (AICD) upon secondary stimulation (212). Furthermore, CD4+ T cells have also been demonstrated to be essential for maintaining conventional memory CD8+ T cell numbers in the absence of secondary challenge (213, 214). Although the exact roles and signaling events carried out by CD4+ T cells remain elusive, they undeniably provide essential services for the optimal generation of CD8+ T cell memory (215). 1.4.5 Conventional CD4+ T cells CD4+ T helper (TH) cells are so named because of their ability to provide supplementary signals to B cells or CD8+ T cells so that they can differentiate into fully functional effector cells. TH cells are divided into two subsets, TH1 and TH2, which are defined by  25 the cytokines they secrete. TH1 cells secrete high levels of IFN-γ and are important for immune responses to intracellular pathogens, whereas TH2 responses, which secrete high levels of IL-4, are specialized in assisting B cells that produce antibodies for defense against extracellular pathogens. In addition to these two subsets, CD4+ T cells that secrete IL-17 (TH17) have recently been identified. TH17 cells are involved in mediating immune responses against extracellular pathogenic bacteria, in inflammatory and autoimmune diseases, and in immune responses against cancer cells (216, 217). In contrast to the ability of these three TH subsets to mediate immune responses by carrying out inflammatory functions, a different subset of CD4+ T cells possess regulatory functions. These regulatory CD4+ T cells play an indispensable role in the maintenance of self-tolerance and immune homeostasis (218). The development of TH1 lineage cells from naïve CD4+ T cells is dependent on recognition of appropriate peptides presented in the context of MHC class II molecules and a source of IL-12 derived from innate sources such as macrophages or DCs. IL-12 activates the STAT4 signaling pathways, activating genes that encode for IFN-γ and the T-box family transcription factor T-bet, which is considered to be the master regulator of TH1 cell differentiation (219). T-bet upregulates the receptor for IL-12 making TH1 cells even more sensitive to this polarizing cytokine (220-223). The importance of this cytokine is clearly exemplified by the diminished TH1 response against several different pathogens in mice deficient in IL-12 (224). However, recent work has identified a key role for the Notch signaling pathway in promoting the differentiation of naïve CD4+ T cells into TH1 cells in the absence of IL-12 (225). In addition to secreting copious amount of IFN-γ that can fully induce the antimicrobial abilities of macrophages,  26 activated TH1 cells express CD40 ligand (CD40L), which when bound to CD40 on the surface of DCs results in the ability of APCs to induce and support the effector functions of CD8+ T cells (226-228). In contrast to TH1 cells, the transcription factor GATA-3 together with IL-4 stimulation leads to the activation of the STAT6 signaling pathway, which results in TH2 differentiation (229, 230). IL-4, the signature molecule of TH2 cells, triggers isotype switching towards immunoglobulin (Ig)E in B cells, which is necessary for combating extracellular parasites. Moreover, GATA-3, considered to be the master regulator of TH2 differentiation, further augments IL-4 production, creating a positive feedback loop and the induction of the expression of other TH2-related cytokines such as IL-5, IL-9, IL-10, IL-13 and IL-25, which play key roles in immune responses against extracellular pathogens (231). The development of TH17 cells from naïve CD4+ T cells is critically dependent on transforming growth factor (TGF)-β, and IL-6 (232). Their development is amplified by IL-21 and stabilized by IL-23 (233). IL-6 and IL-21 activates the STAT3 signaling pathway that leads to the expression of retinoic-acid-receptor-related orphan receptor (ROR)γ or RORα, which drives TH17 development (234-240). In addition to being generated in response to extracellular infections from bacterial or fungal sources (241), they have also been implicated to stimulate a whole array of other cell types, including CD8+ T cells, B cells, NK cells and DCs (242). Furthermore, despite being implicated in the progression of inflammatory diseases such as psoriasis and multiple sclerosis, TH17 cells have also been shown to be anti-inflammatory and can protect against certain conditions through the production of IL-22, which can activate anti-apoptotic responses in tissues (243). Similar to the newly identified regulatory potential of TH17 cells, Treg  27 cells have been studied extensively and demonstrated to be essential for suppressing inappropriate activation of the immune system, which helps to maintain normal immune homeostasis and tolerance to self-Ags (244). Treg cells are mainly produced in the thymus and constitutively express CD25 (IL-2Rα) (245). They also express Foxp3, a key gene important for their development and function, and are actively engaged in the negative control of a variety of physiological and pathological immune responses (246, 247). Activated CD4+CD25+ Treg cells can suppress the proliferation of CD4+ as well as CD8+ T cells by multiple mechanisms that include the secretion of immunosuppressive cytokines, engagement of ligands with suppressive functions, and the elimination of APCs that are driving the T cell response (reviewed in 248).  1.5  The T cell response to Listeria monocytogenes  1.5.1 Overview of Listeria monocytogenes Listeria monocytogenes (LM) is a gram-positive bacterium that can be found in soil, stream water, sewage, plants and food. It is the causative agent of listeriosis, which primarily affects pregnant women and immunocompromised individuals. LM has been greatly characterized and has been widely used as a laboratory model to understand immune responses to intracellular bacterial infections. Upon entering the murine host, the majority of phagocytosed bacteria are initially confined to the phagosome but eventually escape into the macrophage cytosol where they replicate and spread by infecting neighboring cells, which is dependent on the production of Listeriolysin O  28 (LLO) for escape and the ActA protein for motility (249, 250). LM mutants lacking LLO and ActA proteins fail to escape the phagosomal compartment and infect neighboring cells, respectively, and are both relatively nonvirulent (251, 252). In addition to mutant forms of LM, recombinant forms that use LM as a vector to carry different antigenic epitopes for conventional CD8+ T cell also exists. These recombinant LM strains serve as powerful models for studying the mechanism of T cell responses to intracellular bacterial infection. The resistance to LM infection in the murine model requires immune cells from both the innate and adaptive immune systems. Through the use of germline-encoded receptors, innate immune cells such as macrophages, NK cells, and neutrophils rapidly respond in a non-specific manner to limit the initial spread of LM, which provides sufficient time for cells of adaptive immunity to develop (253). Additionally, IFN-γ and TNF-α have been shown to be key cytokines essential for the primary defense against LM infection, since mice lacking these cytokines or their receptors rapidly succumb to infection (253). IFN-γ has been demonstrated to play a key role in mediating LM resistance because of its ability to increase macrophage bactericidal function, which is correlated with the production of reactive nitrogen and oxygen intermediates in macrophages (254). In the event innate immune responses against LM were insufficient to control LM infection, cell-mediated adaptive immune responses are required to provide sterilizing immunity with accompanying long-term immunological protection.  29 1.5.2 MHC class Ia- and Ib-restricted T cell response to LM infection Although CD4+ T cells respond during the course of infection, CD8+ T cells are the principle cell type that provides sterilizing immunity to LM infection. Upon entering eukaryotic cells, LM escapes into the cytosol of the infected cells where secreted bacterial-derived proteins are processed into peptides by the proteasome and subsequently presented by MHC class Ia- and class Ib-restricted molecules to their respective T cells (Figure 1.4). The main APCs that prime these CD8+ T cells are CD11c+-expressing DCs (255). Interestingly, these two T cell populations respond to LM bacterial infections with distinct patterns and kinetics. Specifically, the MHC class Ib molecules, H2-M3, presents LM-derived N-formylated peptides to nonconventional CD8+ T cells resulting in their ability to reach peak frequencies earlier than the MHC class Ia-restricted response during primary infection (days 5-7 vs. days 7-9 after infection) (256, 257). To date, three listerial peptides presented by H2-M3 have been identified, including fMIVIL, fMIVTLF, and fMIGWII, the latter being the immunodominant peptide (91). In addition to a protective role in mice with a fully functional immune system, H2-M3-restricted T cells have also been shown to provide protection by rapidly producing cytokines and directly killing infected cells during both primary and secondary LM infection in mice that lack conventional CD8+ T cells (78, 257, 258). However, despite their prompt and robust primary response, H2-M3restricted T cells fail to undergo significant expansion upon secondary LM infection. By contrast, MHC class Ia-restricted T cells undergo vigorous expansion during secondary LM infection, which provides sterilizing anti-bacterial immunity (256-258). Since H2-M3restricted T cells can provide protection in the absence conventional CD8+ T cells, a  30 plausible explanation to this observation could be that H2-M3-restricted T cells compete inefficiently with MHC class Ia-restricted T cells for APCs during secondary LM immune responses. This notion was supported by a study demonstrating the ability for MHC class Ia-restricted memory T cells to prevent the expansion of H2-M3-restricted T cell populations by limiting DC Ag presentation in mice previously infected with LM (259). Alternatively, this observation may be explained by different localization patterns of T cells subsets and their ability to elicit a kinetically distinct immune response in different organs. Whereas MHC class Ia-restricted memory T cells predominate in spleens following tail-vein secondary infection with LM, H2-M3-restricted T cells remain prominent in lymph nodes draining secondary infections and vigorously respond following tissue infection with LM (260). More importantly, the ability of H2-M3-restricted T cells to become rapidly activated and produce early sources of pro-inflammatory cytokines demonstrate they provide functions that are distinct from conventional CD8+ T cells and suggest that they serve a non-redundant role by functioning as a temporal bridges that link the innate with adaptive immune responses (261). 1.6  Thesis rationale, hypothesis, objectives, and significance  H2-M3-restricted CD8+ T cells share properties with cells of the innate and adaptive immune system. Specifically, they rapidly produce proinflammatory cytokines with innate-like kinetics following their activation by recognizing N-formylated peptides using gene rearranged TCRs. H2-M3-restricted CD8+ T cells are cytolytic and provide immediate protection against infection with LM. Mice deficient for H2-M3 molecules are impaired in early bacterial clearance during primary infection and display compromised innate immune functions, confirming that these molecules perform a non-redundant  31 function in immune responses to bacterial infection. Although they respond vigorously during the primary immune response, their secondary responses to bacterial rechallenge are significantly less robust, which suggests their primary role is likely confined to the early immune response. Although H2-M3-restricted T cells are well known for their protective roles during primary bacterial infections, their ability to influence the developing adaptive immune response has not been determined.  The aim of this thesis is to investigate the potential for H2-M3-restricted CD8+ T cells to shape downstream adaptive immune responses, and to investigate the mechanism used to perform this function. I will test the hypothesis that the activation of H2-M3-restricted CD8+ T cells leads to the rapid generation of pro-inflammatory cytokines that can have pleiotropic effects on dendritic cells, leading to enhanced priming of adaptive T cell immune responses.  The significance of this study is exemplified by demonstrating for the first time, the ability for MHC class Ib-restricted T cell subsets to serve an immunoregulatory function. The results obtained from this study provide a better understanding of the functions of these cells, as well as the mechanism utilized by H2-M3-restricted CD8+ T cells, and potentially other T cells restricted by MHC class Ib molecules, to augment adaptive immune responses. This new discovery may lead to novel approaches in the design of more effective vaccines against microbial infections and possibly cancer immunotherapy, successful responses against cancer is crucially dependent on CD4+ T cell help.  32 The specific aims of this thesis are: (1) to determine if H2-M3-restricted CD8+ T cells can function as immunoregulatory cells, (2) to demonstrate the ability of H2-M3restricted T cells to enhance cell-mediated immune responses, (3) to determine the mechanism used by H2-M3-restricted CD8+ T cells to influence adaptive immune responses, and (4) to determine if other nonconventional CD8+ T cells can function as immunoregulatory cells.  33  Table 1.1  Human and murine MHC class Ia and Ib molecules. Table from Rodgers,  J.R., and Cook, R.G. MHC class Ib molecules bridge innate and acquired immunity. 2005. Nat Rev Immunol. 5(6): 459-71.  34  Table 1.2  Phenotypic and functional differences between dendritic cell subsets.  Table from Liu, Y. 2001. Cell. 3(106): 259-262.  35  Table 1.3  Phenotypic and functional differences between conventional and innate-  like T cells. Table from Berg, L.J. 2007. Nat Rev Immunol. 7(6): 479-85.  36  Figure 1.1  The human and mouse MHC. A simplified diagram of the human MHC on  chromosome 6 is shown in the top part of the figure, with the mouse MHC on chromosome 17 below it. The organization of the mouse and human MHCs is very similar, except that mouse class I genes have become separated at either end of the MHC. This is believed to reflect a chromosomal rearrangement that occurred after the divergence of mouse and human. The gene for 2-microglobulin, which is encoded outside the MHC on human chromosome 15 and mouse chromosome 2, are not shown. C4A, C4B, C2, Bf: genes for complement components; DN, DM, DO, M, O: nonclassical class II genes; DP, DQ, DR, A, E: classical MHC class II genes; HLA-A, HLAB, HLA-C, K, D, L: classical MHC class I genes; LMP: genes for components of the proteasome, the enzyme complex that degrades proteins into peptides in the cytoplasm; LT: genes for lymphotoxin; MICA, MICB, HLA-E, HLA-G, HLA-F, HFE, Q, T, M: genes for nonclassical MHC class I molecules; TAP: genes for the transporter through which peptides from the cytoplasm enter the endoplasmic reticulum; TAPBP: gene for tapasin, a chaperone molecule involved in MHC class I peptide loading; TNF: genes for tumor necrosis factor, an important cytokine. Figure from Maragulies, D.H. et al. 2003. The  37 major histocompatibility complex and its encoded proteins. Fundamental Immunology 5th ed. Paul, W.E. ed. 571-612.  38  Figure 1.2  Overall scheme of T-cell development in the thymus. Committed lymphoid  progenitors arise in the bone marrow and migrate to the thymus. Early committed T cells lack expression of T-cell receptor (TCR), CD4 and CD8, and are termed doublenegative (DN; no CD4 or CD8) thymocytes. DN thymocytes can be further subdivided into four stages of differentiation (DN1, CD44+CD25−; DN2, CD44+CD25+; DN3, CD44−CD25+; and DN4, CD44−CD25−) (189). As cells progress through the DN2 to DN4 stages, they express the pre-TCR, which is composed of the non-rearranging pre-  39 Tα chain and a rearranged TCR β-chain (190). Successful pre-TCR expression leads to substantial cell proliferation during the DN4 to double positive (DP) transition and replacement of the pre-TCR α-chain with a newly rearranged TCR α-chain, which yields a complete αβ TCR. The αβ-TCR+CD4+CD8+ (DP) thymocytes then interact with cortical epithelial cells that express a high density of MHC class I and class II molecules associated with self-peptides. The fate of the DP thymocytes depends on signaling that is mediated by interaction of the TCR with these self-peptide–MHC ligands (191, 192). Too little signaling result in delayed apoptosis (death by neglect), whereas too much signaling promotes acute apoptosis (negative selection); the latter of which is most common in the medulla on encounter with strongly activating self-ligands on hematopoietic cells, particularly dendritic cells (193). The appropriate, intermediate level of TCR signaling initiates effective maturation (positive selection). Thymocytes that express TCRs that bind self peptide–MHC-class-I complexes become CD8+ T cells, whereas those that express TCRs that bind self-peptide–MHC-class-II ligands become CD4+ T cells; these cells are then ready for export from the medulla to peripheral lymphoid sites. SP, single positive. Figure from Germain, R.N. 2002. T-cell development and the CD4-CD8 lineage decision. Nat Rev Immunol 2:309-322. (194).  40  Figure 1.3  Model proposing distinct requirements for conventional versus innate T  cell development in the thymus. The two general types of mature αβ TCR-expressing T cells develop from CD4+CD8+ thymocyte progenitors are conventional naive CD4+ and CD8+ T cells, and innate T cells. The two main features distinguishing the selection of conventional versus innate T cells are differences in TCR specificity and/or affinity, and dependence on interactions with epithelial cells versus bone-marrow-derived cells, such as thymocytes. Conventional naive CD4+ and CD8+ T cells undergo positive selection following moderate-affinity TCR interactions with classical MHC class I or II molecules expressed on thymic epithelial cells. Therefore, the nature of the MHC ligand, the affinity of the TCR for this ligand and putative co-stimulatory molecules expressed on cortical epithelial cells all contribute to the activation of ITK (interleukin-2 (IL-2)-inducible  41 T-cell kinase) and RLK (resting lymphocyte kinase), which lead to downstream signals (currently undefined) that induce the development of conventional naive T cells. By contrast, positive selection resulting from higher-affinity TCR interactions or interactions with non-classical MHC class Ib molecules expressed on thymocytes promotes differentiation into innate T-cell lineages. Co-stimulatory signals that are uniquely provided by thymocytes also contribute to innate T-cell lineage commitment. Together, these signals activate pathways that are ITK and/or RLK-independent, and promote the induction of transcription factors, such as eomesodermin (EOMES) or T-bet, that upregulate memory-cell receptor expression and effector functions, such as the production of effector cytokines. Figure from Berg, L.J. 2007. Nat Rev Immunol. 7(6): 479-85.  42  Figure 1.4  Presentation of Listeria monocytogenes-derived antigens to CD8+ T cells.  Listeria monocytogenes-derived antigens that are presented to CD8+ T cells can be  43 divided into two categories. a | The first type, which are presented by MHC class Ia molecules, are derived by proteasome-mediated degradation of proteins that L. monocytogenes secretes into the host cell cytosol. b | The second type, which are presented by H2–M3 MHC class Ib molecules, are released by L. monocytogenes into the infected cell and consist of short, hydrophobic peptides that initiate with N-formyl methionine (fM). Examples of these are shown. β2m, β2-microglobulin; LLO, listeriolysin O; Mpl, zinc metalloproteinase; p60, murine hydrolase; TAP, transporter associated with antigen processing; TCR, T-cell receptor. Figure from Pamer, E.G. 2004. Nat Rev Immunol. 4(10): 812-23.  44 1.7  1.  References  Urdahl, K. B., J. C. Sun, and M. J. Bevan. 2002. Positive selection of MHC class Ib-restricted CD8(+) T cells on hematopoietic cells. Nature immunology 3:772779.  2.  Pamer, E. G. 2004. Immune responses to Listeria monocytogenes. Nature Reviews Immunology 4:812-823.  3.  Prime, S. S., A. Pitigala-Arachchi, I. J. Crane, T. J. Rosser, and C. Scully. 1987. The expression of cell surface MHC class I heavy and light chain molecules in pre-malignant and malignant lesions of the oral mucosa. Histopathology 11:8191.  4.  Bjorkman, P. J., M. A. Saper, B. Samraoui, W. S. Bennett, J. L. Strominger, and D. C. Wiley. 1987. The foreign antigen binding site and T cell recognition regions of class I histocompatibility antigens. Nature 329:512-518.  5.  Agrawal, S., and M. C. Kishore. 2000. MHC class I gene expression and regulation. J Hematother Stem Cell Res 9:795-812.  6.  Simpson, E. 1988. Function of the MHC. Immunol Suppl 1:27-30.  7.  Potts, W. K., and E. K. Wakeland. 1990. The maintenance of MHC polymorphism. Immunology today 11:39-40.  8.  Shawar, S. M., J. M. Vyas, J. R. Rodgers, and R. R. Rich. 1994. Antigen presentation by major histocompatibility complex class I-B molecules. Annual review of immunology 12:839-880.  45 9.  Parham, P. 1994. The rise and fall of great class I genes. Seminars in immunology 6:373-382.  10.  Neefjes, J. J., F. Momburg, and G. J. Hammerling. 1993. Selective and ATPdependent translocation of peptides by the MHC-encoded transporter. Science (New York, N.Y 261:769-771.  11.  Cresswell, P., N. Bangia, T. Dick, and G. Diedrich. 1999. The nature of the MHC class I peptide loading complex. Immunological reviews 172:21-28.  12.  Robinson, P. J. 1987. Two different biosynthetic pathways for the secretion of Qa region-associated class I antigens by mouse lymphocytes. Proceedings of the National Academy of Sciences of the United States of America 84:527-531.  13.  Brigl, M., and M. B. Brenner. 2004. CD1: antigen presentation and T cell function. Annual review of immunology 22:817-890.  14.  Braud, V. M., D. S. Allan, and A. J. McMichael. 1999. Functions of nonclassical MHC and non-MHC-encoded class I molecules. Current opinion in immunology 11:100-108.  15.  Hansen, T. H., S. Huang, P. L. Arnold, and D. H. Fremont. 2007. Patterns of nonclassical MHC antigen presentation. Nature immunology 8:563-568.  16.  Ehrlich, R., and F. A. Lemonnier. 2000. HFE--a novel nonclassical class I molecule that is involved in iron metabolism. Immunity 13:585-588.  17.  Calabi, F., and C. Milstein. 1986. A novel family of human major histocompatibility complex-related genes not mapping to chromosome 6. Nature 323:540-543.  46 18.  Hughes, A. L., M. Yeager, A. E. Ten Elshof, and M. J. Chorney. 1999. A new taxonomy of mammalian MHC class I molecules. Immunology today 20:22-26.  19.  O'Callaghan, C. A., J. Tormo, B. E. Willcox, V. M. Braud, B. K. Jakobsen, D. I. Stuart, A. J. McMichael, J. I. Bell, and E. Y. Jones. 1998. Structural features impose tight peptide binding specificity in the nonclassical MHC molecule HLA-E. Molecular cell 1:531-541.  20.  Wang, C. R., A. R. Castano, P. A. Peterson, C. Slaughter, K. F. Lindahl, and J. Deisenhofer. 1995. Nonclassical binding of formylated peptide in crystal structure of the MHC class Ib molecule H2-M3. Cell 82:655-664.  21.  Tabaczewski, P., E. Chiang, M. Henson, and I. Stroynowski. 1997. Alternative peptide binding motifs of Qa-2 class Ib molecules define rules for binding of self and nonself peptides. J Immunol 159:2771-2781.  22.  DeCloux, A., A. S. Woods, R. J. Cotter, M. J. Soloski, and J. Forman. 1997. Dominance of a single peptide bound to the class I(B) molecule, Qa-1b. J Immunol 158:2183-2191.  23.  Lindahl, K. F., D. E. Byers, V. M. Dabhi, R. Hovik, E. P. Jones, G. P. Smith, C. R. Wang, H. Xiao, and M. Yoshino. 1997. H2-M3, a full-service class Ib histocompatibility antigen. Annual review of immunology 15:851-879.  24.  Holcombe, H. R., A. R. Castano, H. Cheroutre, M. Teitell, J. K. Maher, P. A. Peterson, and M. Kronenberg. 1995. Nonclassical behavior of the thymus leukemia antigen: peptide transporter-independent expression of a nonclassical class I molecule. The Journal of experimental medicine 181:1433-1443.  47 25.  Liu, Y., Y. Xiong, O. V. Naidenko, J. H. Liu, R. Zhang, A. Joachimiak, M. Kronenberg, H. Cheroutre, E. L. Reinherz, and J. H. Wang. 2003. The crystal structure of a TL/CD8alphaalpha complex at 2.1 A resolution: implications for modulation of T cell activation and memory. Immunity 18:205-215.  26.  Wingren, C., M. P. Crowley, M. Degano, Y. Chien, and I. A. Wilson. 2000. Crystal structure of a gammadelta T cell receptor ligand T22: a truncated MHC-like fold. Science (New York, N.Y 287:310-314.  27.  Loveland, B., C. R. Wang, H. Yonekawa, E. Hermel, and K. F. Lindahl. 1990. Maternally transmitted histocompatibility antigen of mice: a hydrophobic peptide of a mitochondrially encoded protein. Cell 60:971-980.  28.  Shawar, S. M., R. G. Cook, J. R. Rodgers, and R. R. Rich. 1990. Specialized functions of MHC class I molecules. I. An N-formyl peptide receptor is required for construction of the class I antigen Mta. The Journal of experimental medicine 171:897-912.  29.  Rotzschke, O., K. Falk, S. Stevanovic, B. Grahovac, M. J. Soloski, G. Jung, and H. G. Rammensee. 1993. Qa-2 molecules are peptide receptors of higher stringency than ordinary class I molecules. Nature 361:642-644.  30.  Milligan, G. N., L. Flaherty, V. L. Braciale, and T. J. Braciale. 1991. Nonconventional (TL-encoded) major histocompatibility complex molecules present processed viral antigen to cytotoxic T lymphocytes. The Journal of experimental medicine 174:133-138.  31.  Monaco, J. J. 1992. Major histocompatibility complex-linked transport proteins and antigen processing. Immunologic research 11:125-132.  48 32.  Rodgers, J. R., and R. G. Cook. 2005. MHC class Ib molecules bridge innate and acquired immunity. Nature Reviews Immunology 5:459-471.  33.  Sullivan, L. C., H. L. Hoare, J. McCluskey, J. Rossjohn, and A. G. Brooks. 2006. A structural perspective on MHC class Ib molecules in adaptive immunity. Trends in immunology 27:413-420.  34.  Borrego, F., M. Ulbrecht, E. H. Weiss, J. E. Coligan, and A. G. Brooks. 1998. Recognition of human histocompatibility leukocyte antigen (HLA)-E complexed with HLA class I signal sequence-derived peptides by CD94/NKG2 confers protection from natural killer cell-mediated lysis. The Journal of experimental medicine 187:813-818.  35.  Vance, R. E., J. R. Kraft, J. D. Altman, P. E. Jensen, and D. H. Raulet. 1998. Mouse CD94/NKG2A is a natural killer cell receptor for the nonclassical major histocompatibility complex (MHC) class I molecule Qa-1(b). The Journal of experimental medicine 188:1841-1848.  36.  Lee, N., D. R. Goodlett, A. Ishitani, H. Marquardt, and D. E. Geraghty. 1998. HLA-E surface expression depends on binding of TAP-dependent peptides derived from certain HLA class I signal sequences. J Immunol 160:4951-4960.  37.  van der Ven, K., K. Pfeiffer, and S. Skrablin. 2000. HLA-G polymorphisms and molecule function--questions and more questions--a review. Placenta 21 Suppl A:S86-92.  38.  Le Bouteiller, P., C. Solier, J. Proll, M. Aguerre-Girr, S. Fournel, and F. Lenfant. 1999. Placental HLA-G protein expression in vivo: where and what for? Human reproduction update 5:223-233.  49 39.  Le Bouteiller, P., and A. Blaschitz. 1999. The functionality of HLA-G is emerging. Immunological reviews 167:233-244.  40.  Colonna, M., J. Samaridis, M. Cella, L. Angman, R. L. Allen, C. A. O'Callaghan, R. Dunbar, G. S. Ogg, V. Cerundolo, and A. Rolink. 1998. Human myelomonocytic cells express an inhibitory receptor for classical and nonclassical MHC class I molecules. J Immunol 160:3096-3100.  41.  Shiroishi, M., K. Tsumoto, K. Amano, Y. Shirakihara, M. Colonna, V. M. Braud, D. S. Allan, A. Makadzange, S. Rowland-Jones, B. Willcox, E. Y. Jones, P. A. van der Merwe, I. Kumagai, and K. Maenaka. 2003. Human inhibitory receptors Ig-like transcript 2 (ILT2) and ILT4 compete with CD8 for MHC class I binding and bind preferentially to HLA-G. Proceedings of the National Academy of Sciences of the United States of America 100:8856-8861.  42.  Lepin, E. J., J. M. Bastin, D. S. Allan, G. Roncador, V. M. Braud, D. Y. Mason, P. A. van der Merwe, A. J. McMichael, J. I. Bell, S. H. Powis, and C. A. O'Callaghan. 2000. Functional characterization of HLA-F and binding of HLA-F tetramers to ILT2 and ILT4 receptors. European journal of immunology 30:35523561.  43.  Shobu, T., N. Sageshima, H. Tokui, M. Omura, K. Saito, Y. Nagatsuka, M. Nakanishi, Y. Hayashi, K. Hatake, and A. Ishitani. 2006. The surface expression of HLA-F on decidual trophoblasts increases from mid to term gestation. Journal of reproductive immunology 72:18-32.  44.  Stroynowski, I. 1990. Molecules related to class-I major histocompatibility complex antigens. Annual review of immunology 8:501-530.  50 45.  Cook, R. G., B. Leone, J. W. Leone, S. M. Widacki, and P. J. Zavell. 1992. Characterization of T cell proliferative responses induced by anti-Qa-2 monoclonal antibodies. Cellular immunology 144:367-381.  46.  Flaherty, L., E. Elliott, J. A. Tine, A. C. Walsh, and J. B. Waters. 1990. Immunogenetics of the Q and TL regions of the mouse. Critical reviews in immunology 10:131-175.  47.  Eghtesady, P., K. A. Brorson, H. Cheroutre, R. E. Tigelaar, L. Hood, and M. Kronenberg. 1992. Expression of mouse Tla region class I genes in tissues enriched for gamma delta cells. Immunogenetics 36:377-388.  48.  Wang, C. R., and K. F. Lindahl. 1993. Organization and structure of the H-2M4M8 class I genes in the mouse major histocompatibility complex. Immunogenetics 38:258-271.  49.  Pamer, E. G., M. J. Bevan, and K. F. Lindahl. 1993. Do nonclassical, class Ib MHC molecules present bacterial antigens to T cells? Trends in microbiology 1:35-38.  50.  Stroynowski, I., M. Soloski, M. G. Low, and L. Hood. 1987. A single gene encodes soluble and membrane-bound forms of the major histocompatibility Qa2 antigen: anchoring of the product by a phospholipid tail. Cell 50:759-768.  51.  Tabaczewski, P., and I. Stroynowski. 1994. Expression of secreted and glycosylphosphatidylinositol-bound Qa-2 molecules is dependent on functional TAP-2 peptide transporter. J Immunol 152:5268-5274.  51 52.  Tian, H., F. Imani, and M. J. Soloski. 1991. Physical and molecular genetic analysis of Qa-2 antigen expression: multiple factors controlling cell surface levels. Molecular immunology 28:845-854.  53.  Tian, Z., Y. Xu, and C. M. Warner. 1992. Removal of Qa-2 antigen alters the Ped gene phenotype of preimplantation mouse embryos. Biology of reproduction 47:271-276.  54.  Wu, L., H. Feng, and C. M. Warner. 1999. Identification of two major histocompatibility complex class Ib genes, Q7 and Q9, as the Ped gene in the mouse. Biology of reproduction 60:1114-1119.  55.  Chiang, E. Y., and I. Stroynowski. 2004. A nonclassical MHC class I molecule restricts CTL-mediated rejection of a syngeneic melanoma tumor. J Immunol 173:4394-4401.  56.  Zappacosta, F., P. Tabaczewski, K. C. Parker, J. E. Coligan, and I. Stroynowski. 2000. The murine liver-specific nonclassical MHC class I molecule Q10 binds a classical peptide repertoire. J Immunol 164:1906-1915.  57.  Ohtsuka, M., H. Inoko, J. K. Kulski, and S. Yoshimura. 2008. Major histocompatibility complex (Mhc) class Ib gene duplications, organization and expression patterns in mouse strain C57BL/6. BMC genomics 9:178.  58.  Tsujimura, K., Y. Obata, Y. Matsudaira, S. Ozeki, K. Yoshikawa, S. Saga, and T. Takahashi. 2001. The binding of thymus leukemia (TL) antigen tetramers to normal intestinal intraepithelial lymphocytes and thymocytes. J Immunol 167:759764.  52 59.  Tsujimura, K., Y. Obata, E. Kondo, K. Nishida, Y. Matsudaira, Y. Akatsuka, K. Kuzushima, and T. Takahashi. 2003. Thymus leukemia antigen (TL)-specific cytotoxic T lymphocytes recognize the alpha1/alpha2 domain of TL free from antigenic peptides. International immunology 15:1319-1326.  60.  Crowley, M. P., Z. Reich, N. Mavaddat, J. D. Altman, and Y. Chien. 1997. The recognition of the nonclassical major histocompatibility complex (MHC) class I molecule, T10, by the gammadelta T cell, G8. The Journal of experimental medicine 185:1223-1230.  61.  Yeager, M., S. Kumar, and A. L. Hughes. 1997. Sequence convergence in the peptide-binding region of primate and rodent MHC class Ib molecules. Molecular biology and evolution 14:1035-1041.  62.  Houchins, J. P., L. L. Lanier, E. C. Niemi, J. H. Phillips, and J. C. Ryan. 1997. Natural killer cell cytolytic activity is inhibited by NKG2-A and activated by NKG2C. J Immunol 158:3603-3609.  63.  Soloski, M. J., A. DeCloux, C. J. Aldrich, and J. Forman. 1995. Structural and functional characteristics of the class IB molecule, Qa-1. Immunological reviews 147:67-89.  64.  Aldrich, C. J., R. Waltrip, E. Hermel, M. Attaya, K. F. Lindahl, J. J. Monaco, and J. Forman. 1992. T cell recognition of QA-1b antigens on cells lacking a functional Tap-2 transporter. J Immunol 149:3773-3777.  65.  Jiang, H., and L. Chess. 2000. The specific regulation of immune responses by CD8+ T cells restricted by the MHC class Ib molecule, Qa-1. Annual review of immunology 18:185-216.  53 66.  Hu, D., K. Ikizawa, L. Lu, M. E. Sanchirico, M. L. Shinohara, and H. Cantor. 2004. Analysis of regulatory CD8 T cells in Qa-1-deficient mice. Nature immunology 5:516-523.  67.  Sarantopoulos, S., L. Lu, and H. Cantor. 2004. Qa-1 restriction of CD8+ suppressor T cells. The Journal of clinical investigation 114:1218-1221.  68.  Jensen, P. E., B. A. Sullivan, L. M. Reed-Loisel, and D. A. Weber. 2004. Qa-1, a nonclassical class I histocompatibility molecule with roles in innate and adaptive immunity. Immunologic research 29:81-92.  69.  Jiang, H., R. Ware, A. Stall, L. Flaherty, L. Chess, and B. Pernis. 1995. Murine CD8+ T cells that specifically delete autologous CD4+ T cells expressing V beta 8 TCR: a role of the Qa-1 molecule. Immunity 2:185-194.  70.  Lopez-Botet, M., M. Carretero, T. Bellon, J. J. Perez-Villar, M. Llano, and F. Navarro. 1998. The CD94/NKG2 C-type lectin receptor complex. Current topics in microbiology and immunology 230:41-52.  71.  Vance, R. E., A. M. Jamieson, and D. H. Raulet. 1999. Recognition of the class Ib molecule Qa-1(b) by putative activating receptors CD94/NKG2C and CD94/NKG2E on mouse natural killer cells. The Journal of experimental medicine 190:1801-1812.  72.  Long, E. O. 1999. Regulation of immune responses through inhibitory receptors. Annual review of immunology 17:875-904.  73.  Lanier, L. L. 1998. NK cell receptors. Annual review of immunology 16:359-393.  74.  McMahon, C. W., and D. H. Raulet. 2001. Expression and function of NK cell receptors in CD8+ T cells. Current opinion in immunology 13:465-470.  54 75.  Imani, F., and M. J. Soloski. 1991. Heat shock proteins can regulate expression of the Tla region-encoded class Ib molecule Qa-1. Proceedings of the National Academy of Sciences of the United States of America 88:10475-10479.  76.  Lo, W. F., A. S. Woods, A. DeCloux, R. J. Cotter, E. S. Metcalf, and M. J. Soloski. 2000. Molecular mimicry mediated by MHC class Ib molecules after infection with gram-negative pathogens. Nature medicine 6:215-218.  77.  Bouwer, H. G., M. S. Seaman, J. Forman, and D. J. Hinrichs. 1997. MHC class Ib-restricted cells contribute to antilisterial immunity: evidence for Qa-1b as a key restricting element for Listeria-specific CTLs. J Immunol 159:2795-2801.  78.  D'Orazio, S. E., D. G. Halme, H. L. Ploegh, and M. N. Starnbach. 2003. Class Ia MHC-deficient BALB/c mice generate CD8+ T cell-mediated protective immunity against Listeria monocytogenes infection. J Immunol 171:291-298.  79.  Urdahl, K. B., D. Liggitt, and M. J. Bevan. 2003. CD8+ T cells accumulate in the lungs of Mycobacterium tuberculosis-infected Kb-/-Db-/- mice, but provide minimal protection. J Immunol 170:1987-1994.  80.  Smith, G. P., V. M. Dabhi, E. G. Pamer, and K. F. Lindahl. 1994. Peptide presentation by the MHC class Ib molecule, H2-M3. International immunology 6:1917-1926.  81.  Byers, D. E., and K. Fischer Lindahl. 1998. H2-M3 presents a nonformylated viral epitope to CTLs generated in vitro. J Immunol 161:90-96.  82.  Lindahl, K. F., B. Hausmann, and V. M. Chapman. 1983. A new H-2-linked class I gene whose expression depends on a maternally inherited factor. Nature 306:383-385.  55 83.  Wang, C. R., B. E. Loveland, and K. F. Lindahl. 1991. H-2M3 encodes the MHC class I molecule presenting the maternally transmitted antigen of the mouse. Cell 66:335-345.  84.  Vyas, J. M., R. R. Rich, D. D. Howell, S. M. Shawar, and J. R. Rodgers. 1994. Availability of endogenous peptides limits expression of an M3a-Ld major histocompatibility complex class I chimera. The Journal of experimental medicine 179:155-165.  85.  Chiu, N. M., T. Chun, M. Fay, M. Mandal, and C. R. Wang. 1999. The majority of H2-M3 is retained intracellularly in a peptide-receptive state and traffics to the cell surface in the presence of N-formylated peptides. The Journal of experimental medicine 190:423-434.  86.  Lenz, L. L., and M. J. Bevan. 1996. H2-M3 restricted presentation of Listeria monocytogenes antigens. Immunological reviews 151:107-121.  87.  Attaya, M., S. Jameson, C. K. Martinez, E. Hermel, C. Aldrich, J. Forman, K. F. Lindahl, M. J. Bevan, and J. J. Monaco. 1992. Ham-2 corrects the class I antigen-processing defect in RMA-S cells. Nature 355:647-649.  88.  Lenz, L. L., B. Dere, and M. J. Bevan. 1996. Identification of an H2-M3-restricted Listeria epitope: implications for antigen presentation by M3. Immunity 5:63-72.  89.  Gulden, P. H., P. Fischer, 3rd, N. E. Sherman, W. Wang, V. H. Engelhard, J. Shabanowitz, D. F. Hunt, and E. G. Pamer. 1996. A Listeria monocytogenes pentapeptide is presented to cytolytic T lymphocytes by the H2-M3 MHC class Ib molecule. Immunity 5:73-79.  56 90.  Princiotta, M. F., L. L. Lenz, M. J. Bevan, and U. D. Staerz. 1998. H2-M3 restricted presentation of a Listeria-derived leader peptide. The Journal of experimental medicine 187:1711-1719.  91.  Ploss, A., G. Lauvau, B. Contos, K. M. Kerksiek, P. D. Guirnalda, I. Leiner, L. L. Lenz, M. J. Bevan, and E. G. Pamer. 2003. Promiscuity of MHC class Ibrestricted T cell responses. J Immunol 171:5948-5955.  92.  Chun, T., N. V. Serbina, D. Nolt, B. Wang, N. M. Chiu, J. L. Flynn, and C. R. Wang. 2001. Induction of M3-restricted cytotoxic T lymphocyte responses by Nformylated peptides derived from Mycobacterium tuberculosis. The Journal of experimental medicine 193:1213-1220.  93.  Albertson, D. G., R. Fishpool, P. Sherrington, E. Nacheva, and C. Milstein. 1988. Sensitive and high resolution in situ hybridization to human chromosomes using biotin labelled probes: assignment of the human thymocyte CD1 antigen genes to chromosome 1. The EMBO journal 7:2801-2805.  94.  Porcelli, S. A., and R. L. Modlin. 1999. The CD1 system: antigen-presenting molecules for T cell recognition of lipids and glycolipids. Annual review of immunology 17:297-329.  95.  Porcelli, S. A. 1995. The CD1 family: a third lineage of antigen-presenting molecules. Adv Immunol 59:1-98.  96.  van de Rijn, M., P. G. Lerch, B. R. Bronstein, R. W. Knowles, A. K. Bhan, and C. Terhorst. 1984. Human cutaneous dendritic cells express two glycoproteins T6 and M241 which are biochemically identical to those found on cortical thymocytes. Human immunology 9:201-210.  57 97.  Calabi, F., J. M. Jarvis, L. Martin, and C. Milstein. 1989. Two classes of CD1 genes. European journal of immunology 19:285-292.  98.  Murphy, G. F., B. R. Bronstein, R. W. Knowles, and A. K. Bhan. 1985. Ultrastructural documentation of M241 glycoprotein on dendritic and endothelial cells in normal human skin. Laboratory investigation; a journal of technical methods and pathology 52:264-269.  99.  Small, T. N., R. W. Knowles, C. Keever, N. A. Kernan, N. Collins, R. J. O'Reilly, B. Dupont, and N. Flomenberg. 1987. M241 (CD1) expression on B lymphocytes. J Immunol 138:2864-2868.  100.  Delia, D., G. Cattoretti, N. Polli, E. Fontanella, A. Aiello, R. Giardini, F. Rilke, and G. Della Porta. 1988. CD1c but neither CD1a nor CD1b molecules are expressed on normal, activated, and malignant human B cells: identification of a new B-cell subset. Blood 72:241-247.  101.  Porcelli, S., M. B. Brenner, J. L. Greenstein, S. P. Balk, C. Terhorst, and P. A. Bleicher. 1989. Recognition of cluster of differentiation 1 antigens by human CD4-CD8-cytolytic T lymphocytes. Nature 341:447-450.  102.  Canchis, P. W., A. K. Bhan, S. B. Landau, L. Yang, S. P. Balk, and R. S. Blumberg. 1993. Tissue distribution of the non-polymorphic major histocompatibility complex class I-like molecule, CD1d. Immunology 80:561-565.  103.  Wu, D. Y., N. H. Segal, S. Sidobre, M. Kronenberg, and P. B. Chapman. 2003. Cross-presentation of disialoganglioside GD3 to natural killer T cells. The Journal of experimental medicine 198:173-181.  58 104.  Kinjo, Y., and M. Kronenberg. 2005. V alpha14 i NKT cells are innate lymphocytes that participate in the immune response to diverse microbes. Journal of clinical immunology 25:522-533.  105.  Fischer, K., E. Scotet, M. Niemeyer, H. Koebernick, J. Zerrahn, S. Maillet, R. Hurwitz, M. Kursar, M. Bonneville, S. H. Kaufmann, and U. E. Schaible. 2004. Mycobacterial phosphatidylinositol mannoside is a natural antigen for CD1drestricted T cells. Proceedings of the National Academy of Sciences of the United States of America 101:10685-10690.  106.  Zhou, D., J. Mattner, C. Cantu, 3rd, N. Schrantz, N. Yin, Y. Gao, Y. Sagiv, K. Hudspeth, Y. P. Wu, T. Yamashita, S. Teneberg, D. Wang, R. L. Proia, S. B. Levery, P. B. Savage, L. Teyton, and A. Bendelac. 2004. Lysosomal glycosphingolipid recognition by NKT cells. Science (New York, N.Y 306:17861789.  107.  Kronenberg, M., and L. Gapin. 2002. The unconventional lifestyle of NKT cells. Nature Reviews Immunology 2:557-568.  108.  Hermans, I. F., J. D. Silk, U. Gileadi, M. Salio, B. Mathew, G. Ritter, R. Schmidt, A. L. Harris, L. Old, and V. Cerundolo. 2003. NKT cells enhance CD4+ and CD8+ T cell responses to soluble antigen in vivo through direct interaction with dendritic cells. J Immunol 171:5140-5147.  109.  Miyamoto, K., S. Miyake, and T. Yamamura. 2001. A synthetic glycolipid prevents autoimmune encephalomyelitis by inducing TH2 bias of natural killer T cells. Nature 413:531-534.  59 110.  Steinman, R. M. 1991. The dendritic cell system and its role in immunogenicity. Annual review of immunology 9:271-296.  111.  Mellman, I., and R. M. Steinman. 2001. Dendritic cells: specialized and regulated antigen processing machines. Cell 106:255-258.  112.  Vieira, P. L., E. C. de Jong, E. A. Wierenga, M. L. Kapsenberg, and P. Kalinski. 2000. Development of Th1-inducing capacity in myeloid dendritic cells requires environmental instruction. J Immunol 164:4507-4512.  113.  Moser, M., and K. M. Murphy. 2000. Dendritic cell regulation of TH1-TH2 development. Nature immunology 1:199-205.  114.  Vitale, M., M. Della Chiesa, S. Carlomagno, D. Pende, M. Arico, L. Moretta, and A. Moretta. 2005. NK-dependent DC maturation is mediated by TNFalpha and IFNgamma released upon engagement of the NKp30 triggering receptor. Blood 106:566-571.  115.  Hawiger, D., K. Inaba, Y. Dorsett, M. Guo, K. Mahnke, M. Rivera, J. V. Ravetch, R. M. Steinman, and M. C. Nussenzweig. 2001. Dendritic cells induce peripheral T cell unresponsiveness under steady state conditions in vivo. The Journal of experimental medicine 194:769-779.  116.  Bonifaz, L., D. Bonnyay, K. Mahnke, M. Rivera, M. C. Nussenzweig, and R. M. Steinman. 2002. Efficient targeting of protein antigen to the dendritic cell receptor DEC-205 in the steady state leads to antigen presentation on major histocompatibility complex class I products and peripheral CD8+ T cell tolerance. The Journal of experimental medicine 196:1627-1638.  60 117.  Cella, M., F. Sallusto, and A. Lanzavecchia. 1997. Origin, maturation and antigen presenting function of dendritic cells. Current opinion in immunology 9:10-16.  118.  Pulendran, B., J. Banchereau, E. Maraskovsky, and C. Maliszewski. 2001. Modulating the immune response with dendritic cells and their growth factors. Trends in immunology 22:41-47.  119.  D'Amico, A., and L. Wu. 2003. The early progenitors of mouse dendritic cells and plasmacytoid predendritic cells are within the bone marrow hemopoietic precursors expressing Flt3. The Journal of experimental medicine 198:293-303.  120.  Shigematsu, H., B. Reizis, H. Iwasaki, S. Mizuno, D. Hu, D. Traver, P. Leder, N. Sakaguchi, and K. Akashi. 2004. Plasmacytoid dendritic cells activate lymphoidspecific genetic programs irrespective of their cellular origin. Immunity 21:43-53.  121.  Karsunky, H., M. Merad, I. Mende, M. G. Manz, E. G. Engleman, and I. L. Weissman. 2005. Developmental origin of interferon-alpha-producing dendritic cells from hematopoietic precursors. Experimental hematology 33:173-181.  122.  Yang, G. X., Z. X. Lian, K. Kikuchi, Y. Moritoki, A. A. Ansari, Y. J. Liu, S. Ikehara, and M. E. Gershwin. 2005. Plasmacytoid dendritic cells of different origins have distinct characteristics and function: studies of lymphoid progenitors versus myeloid progenitors. J Immunol 175:7281-7287.  123.  Traver, D., K. Akashi, M. Manz, M. Merad, T. Miyamoto, E. G. Engleman, and I. L. Weissman. 2000. Development of CD8alpha-positive dendritic cells from a common myeloid progenitor. Science (New York, N.Y 290:2152-2154.  61 124.  Akashi, K., D. Traver, T. Miyamoto, and I. L. Weissman. 2000. A clonogenic common myeloid progenitor that gives rise to all myeloid lineages. Nature 404:193-197.  125.  Steinman, R. M., M. Pack, and K. Inaba. 1997. Dendritic cells in the T-cell areas of lymphoid organs. Immunological reviews 156:25-37.  126.  Iwasaki, A., and B. L. Kelsall. 1999. Freshly isolated Peyer's patch, but not spleen, dendritic cells produce interleukin 10 and induce the differentiation of T helper type 2 cells. The Journal of experimental medicine 190:229-239.  127.  Wu, L., C. L. Li, and K. Shortman. 1996. Thymic dendritic cell precursors: relationship to the T lymphocyte lineage and phenotype of the dendritic cell progeny. The Journal of experimental medicine 184:903-911.  128.  Vremec, D., and K. Shortman. 1997. Dendritic cell subtypes in mouse lymphoid organs: cross-correlation of surface markers, changes with incubation, and differences among thymus, spleen, and lymph nodes. J Immunol 159:565-573.  129.  Pulendran, B., J. Lingappa, M. K. Kennedy, J. Smith, M. Teepe, A. Rudensky, C. R. Maliszewski, and E. Maraskovsky. 1997. Developmental pathways of dendritic cells in vivo: distinct function, phenotype, and localization of dendritic cell subsets in FLT3 ligand-treated mice. J Immunol 159:2222-2231.  130.  Leenen, P. J., K. Radosevic, J. S. Voerman, B. Salomon, N. van Rooijen, D. Klatzmann, and W. van Ewijk. 1998. Heterogeneity of mouse spleen dendritic cells: in vivo phagocytic activity, expression of macrophage markers, and subpopulation turnover. J Immunol 160:2166-2173.  62 131.  Kronin, V., K. Winkel, G. Suss, A. Kelso, W. Heath, J. Kirberg, H. von Boehmer, and K. Shortman. 1996. A subclass of dendritic cells regulates the response of naive CD8 T cells by limiting their IL-2 production. J Immunol 157:3819-3827.  132.  Suss, G., and K. Shortman. 1996. A subclass of dendritic cells kills CD4 T cells via Fas/Fas-ligand-induced apoptosis. The Journal of experimental medicine 183:1789-1796.  133.  Maldonado-Lopez, R., T. De Smedt, P. Michel, J. Godfroid, B. Pajak, C. Heirman, K. Thielemans, O. Leo, J. Urbain, and M. Moser. 1999. CD8alpha+ and CD8alpha- subclasses of dendritic cells direct the development of distinct T helper cells in vivo. The Journal of experimental medicine 189:587-592.  134.  Pulendran, B., J. L. Smith, G. Caspary, K. Brasel, D. Pettit, E. Maraskovsky, and C. R. Maliszewski. 1999. Distinct dendritic cell subsets differentially regulate the class of immune response in vivo. Proceedings of the National Academy of Sciences of the United States of America 96:1036-1041.  135.  Brasel, K., T. De Smedt, J. L. Smith, and C. R. Maliszewski. 2000. Generation of murine dendritic cells from flt3-ligand-supplemented bone marrow cultures. Blood 96:3029-3039.  136.  Maraskovsky, E., K. Brasel, M. Teepe, E. R. Roux, S. D. Lyman, K. Shortman, and H. J. McKenna. 1996. Dramatic increase in the numbers of functionally mature dendritic cells in Flt3 ligand-treated mice: multiple dendritic cell subpopulations identified. The Journal of experimental medicine 184:1953-1962.  137.  Inaba, K., M. Inaba, N. Romani, H. Aya, M. Deguchi, S. Ikehara, S. Muramatsu, and R. M. Steinman. 1992. Generation of large numbers of dendritic cells from  63 mouse bone marrow cultures supplemented with granulocyte/macrophage colony-stimulating factor. The Journal of experimental medicine 176:1693-1702. 138.  Lutz, M. B., N. Kukutsch, A. L. Ogilvie, S. Rossner, F. Koch, N. Romani, and G. Schuler. 1999. An advanced culture method for generating large quantities of highly pure dendritic cells from mouse bone marrow. Journal of immunological methods 223:77-92.  139.  Saunders, D., K. Lucas, J. Ismaili, L. Wu, E. Maraskovsky, A. Dunn, and K. Shortman. 1996. Dendritic cell development in culture from thymic precursor cells in the absence of granulocyte/macrophage colony-stimulating factor. The Journal of experimental medicine 184:2185-2196.  140.  Lutz, M. B. 2004. IL-3 in dendritic cell development and function: a comparison with GM-CSF and IL-4. Immunobiology 209:79-87.  141.  Janeway, C. A., Jr. 1989. Approaching the asymptote? Evolution and revolution in immunology. Cold Spring Harbor symposia on quantitative biology 54 Pt 1:113.  142.  Akira, S. 2003. Mammalian Toll-like receptors. Current opinion in immunology 15:5-11.  143.  Sallusto, F., and A. Lanzavecchia. 1994. Efficient presentation of soluble antigen by cultured human dendritic cells is maintained by granulocyte/macrophage colony-stimulating factor plus interleukin 4 and downregulated by tumor necrosis factor alpha. The Journal of experimental medicine 179:1109-1118.  144.  Winzler, C., P. Rovere, M. Rescigno, F. Granucci, G. Penna, L. Adorini, V. S. Zimmermann, J. Davoust, and P. Ricciardi-Castagnoli. 1997. Maturation stages  64 of mouse dendritic cells in growth factor-dependent long-term cultures. The Journal of experimental medicine 185:317-328. 145.  Gallucci, S., M. Lolkema, and P. Matzinger. 1999. Natural adjuvants: endogenous activators of dendritic cells. Nature medicine 5:1249-1255.  146.  Luft, T., K. C. Pang, E. Thomas, P. Hertzog, D. N. Hart, J. Trapani, and J. Cebon. 1998. Type I IFNs enhance the terminal differentiation of dendritic cells. J Immunol 161:1947-1953.  147.  Le Bon, A., G. Schiavoni, G. D'Agostino, I. Gresser, F. Belardelli, and D. F. Tough. 2001. Type i interferons potently enhance humoral immunity and can promote isotype switching by stimulating dendritic cells in vivo. Immunity 14:461470.  148.  Sporri, R., and C. Reis e Sousa. 2005. Inflammatory mediators are insufficient for full dendritic cell activation and promote expansion of CD4+ T cell populations lacking helper function. Nature immunology 6:163-170.  149.  Sallusto, F., P. Schaerli, P. Loetscher, C. Schaniel, D. Lenig, C. R. Mackay, S. Qin, and A. Lanzavecchia. 1998. Rapid and coordinated switch in chemokine receptor expression during dendritic cell maturation. European journal of immunology 28:2760-2769.  150.  Matloubian, M., A. David, S. Engel, J. E. Ryan, and J. G. Cyster. 2000. A transmembrane CXC chemokine is a ligand for HIV-coreceptor Bonzo. Nature immunology 1:298-304.  151.  Sozzani, S., P. Allavena, G. D'Amico, W. Luini, G. Bianchi, M. Kataura, T. Imai, O. Yoshie, R. Bonecchi, and A. Mantovani. 1998. Differential regulation of  65 chemokine receptors during dendritic cell maturation: a model for their trafficking properties. J Immunol 161:1083-1086. 152.  Geijtenbeek, T. B., R. Torensma, S. J. van Vliet, G. C. van Duijnhoven, G. J. Adema, Y. van Kooyk, and C. G. Figdor. 2000. Identification of DC-SIGN, a novel dendritic cell-specific ICAM-3 receptor that supports primary immune responses. Cell 100:575-585.  153.  Scheeren, R. A., G. Koopman, S. Van der Baan, C. J. Meijer, and S. T. Pals. 1991. Adhesion receptors involved in clustering of blood dendritic cells and T lymphocytes. European journal of immunology 21:1101-1105.  154.  Lanzavecchia, A., and F. Sallusto. 2000. Dynamics of T lymphocyte responses: intermediates, effectors, and memory cells. Science (New York, N.Y 290:92-97.  155.  Liu, Y. J., H. Kanzler, V. Soumelis, and M. Gilliet. 2001. Dendritic cell lineage, plasticity and cross-regulation. Nature immunology 2:585-589.  156.  Kapsenberg, M. L., C. M. Hilkens, E. A. Wierenga, and P. Kalinski. 1999. The paradigm of type 1 and type 2 antigen-presenting cells. Implications for atopic allergy. Clin Exp Allergy 29 Suppl 2:33-36.  157.  Morelli, A. E., A. F. Zahorchak, A. T. Larregina, B. L. Colvin, A. J. Logar, T. Takayama, L. D. Falo, and A. W. Thomson. 2001. Cytokine production by mouse myeloid dendritic cells in relation to differentiation and terminal maturation induced by lipopolysaccharide or CD40 ligation. Blood 98:1512-1523.  158.  Arase, H., N. Arase, K. Ogasawara, R. A. Good, and K. Onoe. 1992. An NK1.1+ CD4+8- single-positive thymocyte subpopulation that expresses a highly skewed  66 T-cell antigen receptor V beta family. Proceedings of the National Academy of Sciences of the United States of America 89:6506-6510. 159.  Bendelac, A., M. Bonneville, and J. F. Kearney. 2001. Autoreactivity by design: innate B and T lymphocytes. Nature Reviews Immunology 1:177-186.  160.  Rifa'i, M., Y. Kawamoto, I. Nakashima, and H. Suzuki. 2004. Essential roles of CD8+CD122+ regulatory T cells in the maintenance of T cell homeostasis. The Journal of experimental medicine 200:1123-1134.  161.  Endharti, A. T., I. M. Rifa, Z. Shi, Y. Fukuoka, Y. Nakahara, Y. Kawamoto, K. Takeda, K. Isobe, and H. Suzuki. 2005. Cutting edge: CD8+CD122+ regulatory T cells produce IL-10 to suppress IFN-gamma production and proliferation of CD8+ T cells. J Immunol 175:7093-7097.  162.  Akbari, O., P. Stock, E. Meyer, M. Kronenberg, S. Sidobre, T. Nakayama, M. Taniguchi, M. J. Grusby, R. H. DeKruyff, and D. T. Umetsu. 2003. Essential role of NKT cells producing IL-4 and IL-13 in the development of allergen-induced airway hyperreactivity. Nature medicine 9:582-588.  163.  Ikehara, Y., Y. Yasunami, S. Kodama, T. Maki, M. Nakano, T. Nakayama, M. Taniguchi, and S. Ikeda. 2000. CD4(+) Valpha14 natural killer T cells are essential for acceptance of rat islet xenografts in mice. The Journal of clinical investigation 105:1761-1767.  164.  Smyth, M. J., K. Y. Thia, S. E. Street, E. Cretney, J. A. Trapani, M. Taniguchi, T. Kawano, S. B. Pelikan, N. Y. Crowe, and D. I. Godfrey. 2000. Differential tumor surveillance by natural killer (NK) and NKT cells. The Journal of experimental medicine 191:661-668.  67 165.  Wilson, S. B., S. C. Kent, K. T. Patton, T. Orban, R. A. Jackson, M. Exley, S. Porcelli, D. A. Schatz, M. A. Atkinson, S. P. Balk, J. L. Strominger, and D. A. Hafler. 1998. Extreme Th1 bias of invariant Valpha24JalphaQ T cells in type 1 diabetes. Nature 391:177-181.  166.  Chow, M. T., S. Dhanji, J. Cross, P. Johnson, and H. S. Teh. 2006. H2-M3restricted T cells participate in the priming of antigen-specific CD4+ T cells. J Immunol 177:5098-5104.  167.  Perarnau, B., M. F. Saron, B. R. San Martin, N. Bervas, H. Ong, M. J. Soloski, A. G. Smith, J. M. Ure, J. E. Gairin, and F. A. Lemonnier. 1999. Single H2Kb, H2Db and double H2KbDb knockout mice: peripheral CD8+ T cell repertoire and antilymphocytic choriomeningitis virus cytolytic responses. European journal of immunology 29:1243-1252.  168.  Lind, E. F., S. E. Prockop, H. E. Porritt, and H. T. Petrie. 2001. Mapping precursor movement through the postnatal thymus reveals specific microenvironments supporting defined stages of early lymphoid development. The Journal of experimental medicine 194:127-134.  169.  Ardavin, C., L. Wu, C. L. Li, and K. Shortman. 1993. Thymic dendritic cells and T cells develop simultaneously in the thymus from a common precursor population. Nature 362:761-763.  170.  Wu, L., M. Antica, G. R. Johnson, R. Scollay, and K. Shortman. 1991. Developmental potential of the earliest precursor cells from the adult mouse thymus. The Journal of experimental medicine 174:1617-1627.  68 171.  Petrie, H. T., R. Scollay, and K. Shortman. 1992. Commitment to the T cell receptor-alpha beta or -gamma delta lineages can occur just prior to the onset of CD4 and CD8 expression among immature thymocytes. European journal of immunology 22:2185-2188.  172.  Starr, T. K., S. C. Jameson, and K. A. Hogquist. 2003. Positive and negative selection of T cells. Annual review of immunology 21:139-176.  173.  Berg, L. J., and J. Kang. 2001. Molecular determinants of TCR expression and selection. Current opinion in immunology 13:232-241.  174.  Rincon, M., A. Whitmarsh, D. D. Yang, L. Weiss, B. Derijard, P. Jayaraj, R. J. Davis, and R. A. Flavell. 1998. The JNK pathway regulates the In vivo deletion of immature CD4(+)CD8(+) thymocytes. The Journal of experimental medicine 188:1817-1830.  175.  Sugawara, T., T. Moriguchi, E. Nishida, and Y. Takahama. 1998. Differential roles of ERK and p38 MAP kinase pathways in positive and negative selection of T lymphocytes. Immunity 9:565-574.  176.  Basson, M. A., U. Bommhardt, P. J. Mee, V. L. Tybulewicz, and R. Zamoyska. 1998. Molecular requirements for lineage commitment in the thymus--antibodymediated receptor engagements reveal a central role for lck in lineage decisions. Immunological reviews 165:181-194.  177.  Hernandez-Hoyos, G., M. K. Anderson, C. Wang, E. V. Rothenberg, and J. Alberola-Ila. 2003. GATA-3 expression is controlled by TCR signals and regulates CD4/CD8 differentiation. Immunity 19:83-94.  69 178.  Priatel, J. J., X. Chen, S. Dhanji, N. Abraham, and H. S. Teh. 2006. RasGRP1 transmits prodifferentiation TCR signaling that is crucial for CD4 T cell development. J Immunol 177:1470-1480.  179.  He, X., X. He, V. P. Dave, Y. Zhang, X. Hua, E. Nicolas, W. Xu, B. A. Roe, and D. J. Kappes. 2005. The zinc finger transcription factor Th-POK regulates CD4 versus CD8 T-cell lineage commitment. Nature 433:826-833.  180.  Sun, G., X. Liu, P. Mercado, S. R. Jenkinson, M. Kypriotou, L. Feigenbaum, P. Galera, and R. Bosselut. 2005. The zinc finger protein cKrox directs CD4 lineage differentiation during intrathymic T cell positive selection. Nature immunology 6:373-381.  181.  Kappes, D. J., X. He, and X. He. 2005. CD4-CD8 lineage commitment: an inside view. Nature immunology 6:761-766.  182.  Berg, L. J., L. D. Finkelstein, J. A. Lucas, and P. L. Schwartzberg. 2005. Tec family kinases in T lymphocyte development and function. Annual review of immunology 23:549-600.  183.  Chen, X., J. J. Priatel, M. T. Chow, and H. S. Teh. 2008. Preferential development of CD4 and CD8 T regulatory cells in RasGRP1-deficient mice. J Immunol 180:5973-5982.  184.  Atherly, L. O., J. A. Lucas, M. Felices, C. C. Yin, S. L. Reiner, and L. J. Berg. 2006. The Tec family tyrosine kinases Itk and Rlk regulate the development of conventional CD8+ T cells. Immunity 25:79-91.  185.  Baldwin, T. A., K. A. Hogquist, and S. C. Jameson. 2004. The fourth way? Harnessing aggressive tendencies in the thymus. J Immunol 173:6515-6520.  70 186.  Fontenot, J. D., and A. Y. Rudensky. 2005. A well adapted regulatory contrivance: regulatory T cell development and the forkhead family transcription factor Foxp3. Nature immunology 6:331-337.  187.  Cabarrocas, J., C. Cassan, F. Magnusson, E. Piaggio, L. Mars, J. Derbinski, B. Kyewski, D. A. Gross, B. L. Salomon, K. Khazaie, A. Saoudi, and R. S. Liblau. 2006. Foxp3+ CD25+ regulatory T cells specific for a neo-self-antigen develop at the double-positive thymic stage. Proceedings of the National Academy of Sciences of the United States of America 103:8453-8458.  188.  Gapin, L., J. L. Matsuda, C. D. Surh, and M. Kronenberg. 2001. NKT cells derive from double-positive thymocytes that are positively selected by CD1d. Nature immunology 2:971-978.  189.  Chiu, N. M., B. Wang, K. M. Kerksiek, R. Kurlander, E. G. Pamer, and C. R. Wang. 1999. The selection of M3-restricted T cells is dependent on M3 expression and presentation of N-formylated peptides in the thymus. The Journal of experimental medicine 190:1869-1878.  190.  Sullivan, B. A., P. Kraj, D. A. Weber, L. Ignatowicz, and P. E. Jensen. 2002. Positive selection of a Qa-1-restricted T cell receptor with specificity for insulin. Immunity 17:95-105.  191.  Cannarile, M. A., N. Decanis, J. P. van Meerwijk, and T. Brocker. 2004. The role of dendritic cells in selection of classical and nonclassical CD8+ T cells in vivo. J Immunol 173:4799-4805.  192.  Dhanji, S., M. T. Chow, and H. S. Teh. 2006. Self-antigen maintains the innate antibacterial function of self-specific CD8 T cells in vivo. J Immunol 177:138-146.  71 193.  Kawachi, I., J. Maldonado, C. Strader, and S. Gilfillan. 2006. MR1-restricted V alpha 19i mucosal-associated invariant T cells are innate T cells in the gut lamina propria that provide a rapid and diverse cytokine response. J Immunol 176:16181627.  194.  Das, G., S. Sheridan, and C. A. Janeway, Jr. 2001. The source of early IFNgamma that plays a role in Th1 priming. J Immunol 167:2004-2010.  195.  Bendelac, A., M. N. Rivera, S. H. Park, and J. H. Roark. 1997. Mouse CD1specific NK1 T cells: development, specificity, and function. Annual review of immunology 15:535-562.  196.  Townsend, M. J., A. S. Weinmann, J. L. Matsuda, R. Salomon, P. J. Farnham, C. A. Biron, L. Gapin, and L. H. Glimcher. 2004. T-bet regulates the terminal maturation and homeostasis of NK and Valpha14i NKT cells. Immunity 20:477494.  197.  Dubois, S., T. A. Waldmann, and J. R. Muller. 2006. ITK and IL-15 support two distinct subsets of CD8+ T cells. Proceedings of the National Academy of Sciences of the United States of America 103:12075-12080.  198.  Dutton, R. W., L. M. Bradley, and S. L. Swain. 1998. T cell memory. Annual review of immunology 16:201-223.  199.  Ohteki, T. 2002. Critical role for IL-15 in innate immunity. Current molecular medicine 2:371-380.  200.  Linsley, P. S., and J. A. Ledbetter. 1993. The role of the CD28 receptor during T cell responses to antigen. Annual review of immunology 11:191-212.  72 201.  Jameson, S. C. 2002. Maintaining the norm: T-cell homeostasis. Nature Reviews Immunology 2:547-556.  202.  Kaech, S. M., E. J. Wherry, and R. Ahmed. 2002. Effector and memory T-cell differentiation: implications for vaccine development. Nature Reviews Immunology 2:251-262.  203.  Badovinac, V. P., and J. T. Harty. 2006. Programming, demarcating, and manipulating CD8+ T-cell memory. Immunological reviews 211:67-80.  204.  Homann, D., L. Teyton, and M. B. Oldstone. 2001. Differential regulation of antiviral T-cell immunity results in stable CD8+ but declining CD4+ T-cell memory. Nature medicine 7:913-919.  205.  Sallusto, F., D. Lenig, R. Forster, M. Lipp, and A. Lanzavecchia. 1999. Two subsets of memory T lymphocytes with distinct homing potentials and effector functions. Nature 401:708-712.  206.  Weninger, W., M. A. Crowley, N. Manjunath, and U. H. von Andrian. 2001. Migratory properties of naive, effector, and memory CD8(+) T cells. The Journal of experimental medicine 194:953-966.  207.  Wolint, P., M. R. Betts, R. A. Koup, and A. Oxenius. 2004. Immediate cytotoxicity but not degranulation distinguishes effector and memory subsets of CD8+ T cells. The Journal of experimental medicine 199:925-936.  208.  Mullbacher, A., and K. Flynn. 1996. Aspects of cytotoxic T cell memory. Immunological reviews 150:113-127.  73 209.  Becker, T. C., E. J. Wherry, D. Boone, K. Murali-Krishna, R. Antia, A. Ma, and R. Ahmed. 2002. Interleukin 15 is required for proliferative renewal of virus-specific memory CD8 T cells. The Journal of experimental medicine 195:1541-1548.  210.  Goldrath, A. W., P. V. Sivakumar, M. Glaccum, M. K. Kennedy, M. J. Bevan, C. Benoist, D. Mathis, and E. A. Butz. 2002. Cytokine requirements for acute and Basal homeostatic proliferation of naive and memory CD8+ T cells. The Journal of experimental medicine 195:1515-1522.  211.  Schluns, K. S., W. C. Kieper, S. C. Jameson, and L. Lefrancois. 2000. Interleukin-7 mediates the homeostasis of naive and memory CD8 T cells in vivo. Nature immunology 1:426-432.  212.  Janssen, E. M., N. M. Droin, E. E. Lemmens, M. J. Pinkoski, S. J. Bensinger, B. D. Ehst, T. S. Griffith, D. R. Green, and S. P. Schoenberger. 2005. CD4+ T-cell help controls CD8+ T-cell memory via TRAIL-mediated activation-induced cell death. Nature 434:88-93.  213.  Williams, M. A., B. J. Holmes, J. C. Sun, and M. J. Bevan. 2006. Developing and maintaining protective CD8+ memory T cells. Immunological reviews 211:146153.  214.  Sun, J. C., M. A. Williams, and M. J. Bevan. 2004. CD4+ T cells are required for the maintenance, not programming, of memory CD8+ T cells after acute infection. Nature immunology 5:927-933.  215.  Harty, J. T., and V. P. Badovinac. 2008. Shaping and reshaping CD8+ T-cell memory. Nature Reviews Immunology 8:107-119.  74 216.  Ye, P., F. H. Rodriguez, S. Kanaly, K. L. Stocking, J. Schurr, P. Schwarzenberger, P. Oliver, W. Huang, P. Zhang, J. Zhang, J. E. Shellito, G. J. Bagby, S. Nelson, K. Charrier, J. J. Peschon, and J. K. Kolls. 2001. Requirement of interleukin 17 receptor signaling for lung CXC chemokine and granulocyte colony-stimulating factor expression, neutrophil recruitment, and host defense. The Journal of experimental medicine 194:519-527.  217.  Kolls, J. K., and A. Linden. 2004. Interleukin-17 family members and inflammation. Immunity 21:467-476.  218.  Sakaguchi, S., T. Yamaguchi, T. Nomura, and M. Ono. 2008. Regulatory T cells and immune tolerance. Cell 133:775-787.  219.  Djuretic, I. M., D. Levanon, V. Negreanu, Y. Groner, A. Rao, and K. M. Ansel. 2007. Transcription factors T-bet and Runx3 cooperate to activate Ifng and silence Il4 in T helper type 1 cells. Nature immunology 8:145-153.  220.  Hsieh, C. S., S. E. Macatonia, C. S. Tripp, S. F. Wolf, A. O'Garra, and K. M. Murphy. 1993. Development of TH1 CD4+ T cells through IL-12 produced by Listeria-induced macrophages. Science (New York, N.Y 260:547-549.  221.  Hsieh, C. S., S. E. Macatonia, C. S. Tripp, S. F. Wolf, A. O'Garra, and K. M. Murphy. 2008. Pillars article: development of TH1 CD4+ T cells through IL-12 produced by Listeria-induced macrophages. 1993. Science 260(5107): 547-549. J Immunol 181:4437-4439.  222.  Mullen, A. C., F. A. High, A. S. Hutchins, H. W. Lee, A. V. Villarino, D. M. Livingston, A. L. Kung, N. Cereb, T. P. Yao, S. Y. Yang, and S. L. Reiner. 2001.  75 Role of T-bet in commitment of TH1 cells before IL-12-dependent selection. Science (New York, N.Y 292:1907-1910. 223.  Szabo, S. J., B. M. Sullivan, C. Stemmann, A. R. Satoskar, B. P. Sleckman, and L. H. Glimcher. 2002. Distinct effects of T-bet in TH1 lineage commitment and IFN-gamma production in CD4 and CD8 T cells. Science (New York, N.Y 295:338-342.  224.  Magram, J., S. E. Connaughton, R. R. Warrier, D. M. Carvajal, C. Y. Wu, J. Ferrante, C. Stewart, U. Sarmiento, D. A. Faherty, and M. K. Gately. 1996. IL-12deficient mice are defective in IFN gamma production and type 1 cytokine responses. Immunity 4:471-481.  225.  Amsen, D., A. Antov, and R. A. Flavell. 2009. The different faces of Notch in Thelper-cell differentiation. Nature Reviews Immunology 9:116-124.  226.  Ridge, J. P., F. Di Rosa, and P. Matzinger. 1998. A conditioned dendritic cell can be a temporal bridge between a CD4+ T-helper and a T-killer cell. Nature 393:474-478.  227.  Schoenberger, S. P., R. E. Toes, E. I. van der Voort, R. Offringa, and C. J. Melief. 1998. T-cell help for cytotoxic T lymphocytes is mediated by CD40-CD40L interactions. Nature 393:480-483.  228.  Bennett, S. R., F. R. Carbone, F. Karamalis, R. A. Flavell, J. F. Miller, and W. R. Heath. 1998. Help for cytotoxic-T-cell responses is mediated by CD40 signalling. Nature 393:478-480.  76 229.  Ho, I. C., M. R. Hodge, J. W. Rooney, and L. H. Glimcher. 1996. The protooncogene c-maf is responsible for tissue-specific expression of interleukin-4. Cell 85:973-983.  230.  Zhu, J., L. Guo, B. Min, C. J. Watson, J. Hu-Li, H. A. Young, P. N. Tsichlis, and W. E. Paul. 2002. Growth factor independent-1 induced by IL-4 regulates Th2 cell proliferation. Immunity 16:733-744.  231.  Zheng, W., and R. A. Flavell. 1997. The transcription factor GATA-3 is necessary and sufficient for Th2 cytokine gene expression in CD4 T cells. Cell 89:587-596.  232.  Kimura, A., T. Naka, and T. Kishimoto. 2007. IL-6-dependent and -independent pathways in the development of interleukin 17-producing T helper cells. Proceedings of the National Academy of Sciences of the United States of America 104:12099-12104.  233.  Wei, L., A. Laurence, K. M. Elias, and J. J. O'Shea. 2007. IL-21 is produced by Th17 cells and drives IL-17 production in a STAT3-dependent manner. The Journal of biological chemistry 282:34605-34610.  234.  Nurieva, R., X. O. Yang, G. Martinez, Y. Zhang, A. D. Panopoulos, L. Ma, K. Schluns, Q. Tian, S. S. Watowich, A. M. Jetten, and C. Dong. 2007. Essential autocrine regulation by IL-21 in the generation of inflammatory T cells. Nature 448:480-483.  235.  Veldhoen, M., R. J. Hocking, C. J. Atkins, R. M. Locksley, and B. Stockinger. 2006. TGFbeta in the context of an inflammatory cytokine milieu supports de novo differentiation of IL-17-producing T cells. Immunity 24:179-189.  77 236.  Yang, L., D. E. Anderson, C. Baecher-Allan, W. D. Hastings, E. Bettelli, M. Oukka, V. K. Kuchroo, and D. A. Hafler. 2008. IL-21 and TGF-beta are required for differentiation of human T(H)17 cells. Nature 454:350-352.  237.  Mangan, P. R., L. E. Harrington, D. B. O'Quinn, W. S. Helms, D. C. Bullard, C. O. Elson, R. D. Hatton, S. M. Wahl, T. R. Schoeb, and C. T. Weaver. 2006. Transforming growth factor-beta induces development of the T(H)17 lineage. Nature 441:231-234.  238.  Laurence, A., C. M. Tato, T. S. Davidson, Y. Kanno, Z. Chen, Z. Yao, R. B. Blank, F. Meylan, R. Siegel, L. Hennighausen, E. M. Shevach, and J. O'Shea J. 2007. Interleukin-2 signaling via STAT5 constrains T helper 17 cell generation. Immunity 26:371-381.  239.  Ivanov, II, B. S. McKenzie, L. Zhou, C. E. Tadokoro, A. Lepelley, J. J. Lafaille, D. J. Cua, and D. R. Littman. 2006. The orphan nuclear receptor RORgammat directs the differentiation program of proinflammatory IL-17+ T helper cells. Cell 126:1121-1133.  240.  Yang, X. O., B. P. Pappu, R. Nurieva, A. Akimzhanov, H. S. Kang, Y. Chung, L. Ma, B. Shah, A. D. Panopoulos, K. S. Schluns, S. S. Watowich, Q. Tian, A. M. Jetten, and C. Dong. 2008. T helper 17 lineage differentiation is programmed by orphan nuclear receptors ROR alpha and ROR gamma. Immunity 28:29-39.  241.  LeibundGut-Landmann, S., O. Gross, M. J. Robinson, F. Osorio, E. C. Slack, S. V. Tsoni, E. Schweighoffer, V. Tybulewicz, G. D. Brown, J. Ruland, and C. Reis e Sousa. 2007. Syk- and CARD9-dependent coupling of innate immunity to the  78 induction of T helper cells that produce interleukin 17. Nature immunology 8:630638. 242.  Ghilardi, N., and W. Ouyang. 2007. Targeting the development and effector functions of TH17 cells. Seminars in immunology 19:383-393.  243.  Zenewicz, L. A., G. D. Yancopoulos, D. M. Valenzuela, A. J. Murphy, M. Karow, and R. A. Flavell. 2007. Interleukin-22 but not interleukin-17 provides protection to hepatocytes during acute liver inflammation. Immunity 27:647-659.  244.  Wan, Y. Y., and R. A. Flavell. 2008. TGF-beta and regulatory T cell in immunity and autoimmunity. Journal of clinical immunology 28:647-659.  245.  Sakaguchi, S., N. Sakaguchi, M. Asano, M. Itoh, and M. Toda. 1995. Immunologic self-tolerance maintained by activated T cells expressing IL-2 receptor alpha-chains (CD25). Breakdown of a single mechanism of selftolerance causes various autoimmune diseases. J Immunol 155:1151-1164.  246.  Hori, S., T. Nomura, and S. Sakaguchi. 2003. Control of regulatory T cell development by the transcription factor Foxp3. Science (New York, N.Y 299:1057-1061.  247.  Fontenot, J. D., M. A. Gavin, and A. Y. Rudensky. 2003. Foxp3 programs the development and function of CD4+CD25+ regulatory T cells. Nature immunology 4:330-336.  248.  von Boehmer, H. 2005. Mechanisms of suppression by suppressor T cells. Nature immunology 6:338-344.  249.  Cossart, P., M. F. Vicente, J. Mengaud, F. Baquero, J. C. Perez-Diaz, and P. Berche. 1989. Listeriolysin O is essential for virulence of Listeria monocytogenes:  79 direct evidence obtained by gene complementation. Infection and immunity 57:3629-3636. 250.  Dabiri, G. A., J. M. Sanger, D. A. Portnoy, and F. S. Southwick. 1990. Listeria monocytogenes moves rapidly through the host-cell cytoplasm by inducing directional actin assembly. Proceedings of the National Academy of Sciences of the United States of America 87:6068-6072.  251.  Portnoy, D. A., P. S. Jacks, and D. J. Hinrichs. 1988. Role of hemolysin for the intracellular growth of Listeria monocytogenes. The Journal of experimental medicine 167:1459-1471.  252.  Dussurget, O., J. Pizarro-Cerda, and P. Cossart. 2004. Molecular determinants of Listeria monocytogenes virulence. Annual review of microbiology 58:587-610.  253.  Unanue, E. R. 1997. Inter-relationship among macrophages, natural killer cells and neutrophils in early stages of Listeria resistance. Current opinion in immunology 9:35-43.  254.  Chan, J., Y. Xing, R. S. Magliozzo, and B. R. Bloom. 1992. Killing of virulent Mycobacterium tuberculosis by reactive nitrogen intermediates produced by activated murine macrophages. The Journal of experimental medicine 175:11111122.  255.  Jung, S., D. Unutmaz, P. Wong, G. Sano, K. De los Santos, T. Sparwasser, S. Wu, S. Vuthoori, K. Ko, F. Zavala, E. G. Pamer, D. R. Littman, and R. A. Lang. 2002. In vivo depletion of CD11c(+) dendritic cells abrogates priming of CD8(+) T cells by exogenous cell-associated antigens. Immunity 17:211-220.  80 256.  Kerksiek, K. M., D. H. Busch, I. M. Pilip, S. E. Allen, and E. G. Pamer. 1999. H2M3-restricted T cells in bacterial infection: rapid primary but diminished memory responses. The Journal of experimental medicine 190:195-204.  257.  Seaman, M. S., C. R. Wang, and J. Forman. 2000. MHC class Ib-restricted CTL provide protection against primary and secondary Listeria monocytogenes infection. J Immunol 165:5192-5201.  258.  Kerksiek, K. M., A. Ploss, I. Leiner, D. H. Busch, and E. G. Pamer. 2003. H2-M3restricted memory T cells: persistence and activation without expansion. J Immunol 170:1862-1869.  259.  Hamilton, S. E., B. B. Porter, K. A. Messingham, V. P. Badovinac, and J. T. Harty. 2004. MHC class Ia-restricted memory T cells inhibit expansion of a nonprotective MHC class Ib (H2-M3)-restricted memory response. Nature immunology 5:159-168.  260.  Ploss, A., I. Leiner, and E. G. Pamer. 2005. Distinct regulation of H2-M3restricted memory T cell responses in lymph node and spleen. J Immunol 175:5998-6005.  261.  Xu, H., T. Chun, H. J. Choi, B. Wang, and C. R. Wang. 2006. Impaired response to Listeria in H2-M3-deficient mice reveals a nonredundant role of MHC class Ibspecific T cells in host defense. The Journal of experimental medicine 203:449459.  81 Chapter 2  H2-M3-restricted T cells participate in the priming of antigen-specific CD4 T cells 1  2.1 Introduction CD4+ T lymphocytes are at the center of many immune responses because of their multifaceted abilities to support either cell-mediated or humoral immunity. To initiate their program of proliferation into functionally specialized helper cells, they need to be primed by mature dendritic cells (DCs) that have acquired an activated phenotype induced by inflammation, a consequence of innate immune responses against acute infection (1-5). The environment where CD4+ T cell activation takes place, and the interactions with specific innate immune cells, thus has the potential to influence the outcome of the helper T cell response. Innate immune cells respond with rapid kinetics to the presence of pathogens because they express germ-line encoded receptors that recognize non-self molecular patterns (6). The characteristic ability to respond rapidly to conserved pathogen associated molecular patterns are also shared by MHC class Ib molecules (7-10). For instance, H2-M3 molecules present bacterially-derived N-formylated peptides to MHC class Ib-restricted CD8+ T cells (10). Unlike conventional MHC class Ia-restricted CD8+T cells, the H2-M3 response in mice infected with the intracellular bacteria Listeria  1  A version of this chapter has been published. Chow, M.T., Dhanji, S., Cross, J., Johnson, P.,  and Teh, H. S. (2006) H2-M3-restricted T cells participate in the priming of antigen-specific CD4+ T cells. J Immunol 177(8):5098-5104.  82 monocytogenes (LM) peaks several days earlier, which may be due to their activated memory-like phenotype found even in naïve mice (11, 12). Although numerous studies demonstrated the ability for H2-M3-restricted T cells to participate in anti-listerial immunity, there is no evidence to date supporting the notion that H2-M3- restricted T cells can also enhance the activity of conventional CD4+ and CD8+ T cells (11, 13-15). In the present study, we utilized peptide-coated DC immunization to determine the relationship between H2-M3-restricted T cells and Ag-specific CD4+T cells. The results reveal Ag-specific CD4+ T cell responses are enhanced when generated with help from MHC class Ib-restricted T cells, a function conventional CD8+ T cells are unable to perform. Furthermore, we demonstrate the ability of H2-M3-restricted T cells to indirectly augment MHC class Ia-restricted CD8+ T cell responses resulting in a significant increase in protective immunity against primary LM infection. Enhancement in both CD4 and CD8 responses following infection of immune mice also results in greater long-term immunological protection. Moreover, we identified a second MHC class Ib molecule with a human counterpart that is also able to enhance Ag-specific CD4+ T cell responses, thus bridging this system into possible human significance. Our data presented here defines a novel regulatory function for H2-M3- and Qa-1-restricted T cells in strengthening cell-mediated immunity by enhancing Ag-specific CD4+ T cell responses.  83 2.2 Materials and methods  2.2.1 Mice and bacteria C57BL/6 (B6, H-2b) mice were obtained from The Jackson Laboratory (Bar Harbor, ME). Mice 6-12 weeks of age were used for the experiments described. All animal procedures were conducted in accordance with Canadian Council on Animal Care guidelines. The strains of Listeria used in this study have a half-maximal lethal dose of ~ 1 x 105 organisms for B6 mice. The wildtype strain of Listeria 10403s, and a recombinant strain of Listeria XFL203 (referred to as rLM-GP33 in this report) were used; the latter strain expresses the glycoprotein epitope gp33-41 (GP33) from lymphocytic choriomeningitis virus (LCMV) (16). Mice were infected via tail vein inoculation with the number of bacteria indicated in figure legends. 2.2.2 Antibodies and peptides The following mAbs were used: anti-CD4 (GK1.5), anti-CD8α  (53-6.7), anti-CD3ε  (2C11), anti-IFNγ (XMG1.2), and anti-TCRβ (H57-597). All antibodies were obtained from eBioscience (San Diego, CA). The LLO190-201 and GP33-41 peptides were synthesized at the University of British Columbia’s Nucleic Acid-Protein Service Unit. The fMIGWII peptide was synthesized at Sigma Genosys (Oakville, Ontario). 2.2.3 In vivo mAb depletion CD4+ T cells were depleted by i.p. injection of 85μg of anti-CD4 mAb GK1.5 twice at intervals of 3 days beginning 3 days following immunization. Efficacy of depletion in  84 mice treated with GK1.5 mAb in PBS was always >95%, as measured by staining with H57-597, GK1.5 and 53-6.7 mAbs. 2.2.4 Bone marrow-derived dendritic cells Bone marrow-derived CD11c+ DCs were generated by 8 days of culture as described by Lutz et al. (17). The non-adherent cell population consisted of >95% CD11c+ cells following positive selection using the MiniMACS system (Miltenyi Biotec, Auburn, CA) according to the manufacturer’s specifications. For activation and peptide loading of DCs, bone marrow-derived DCs were incubated for 3 hours at 37˚C with 1μg/ml LPS and the indicated peptides in the figures. The DCs were collected following incubation and extensively washed with 1X PBS. Approximately 1 x 106 peptide-coated DCs were injected intravenously via the tail vein. 2.2.5 Detection of antigen-specific T cells The frequency and number of CD4+ and CD8+ T cells specific for LLO190-201 in the context of I-Ab, GP33-41 in the context of H-2Db or fMIG in the context of H2-M3 was determined by intracellular cytokine staining for IFN-γ as described by Hamilton, et al. (18). Synthetic peptides were used at concentration of 2.5μM (fMIG) or 5μM (LLO190-201 or GP33-41). The CellQuest software program (BD Biosciences, Mountain View, CA) was used for data acquisition and analysis.  85 2.3 Results  2.3.1 H2-M3-restricted CD8+T cells participate in the priming of Ag-specific CD4+ T cells To assess the potential influence of MHC class Ib-restricted T cells on the priming of Ag-specific CD4+T cells, mice were immunized with DCs coated with LLO190-201 (LLO) ± the dominant H2-M3-binding formylated peptide found in LM, fMIG (19). The frequency of responding LLO-specific CD4+ and fMIG-specific CD8+ T cells were analyzed 6 days following immunization by restimulating splenocytes with the appropriate peptides and measuring IFN-γ production. Immunization of mice with LLO-coated DCs resulted in the generation of a small but significant population of LLO-specific CD4+ T cells 6 days post immunization. Surprisingly, priming of LLO-specific CD4+ T cells concurrently with H2M3-restricted CD8 T cells, achieved through pulsing the DCs with both LLO and fMIG (LLO + fMIG) peptides, resulted in a 2.2-fold increase in the frequency (Figure 2.1A) of LLO-specific CD4+T cells, which translated to a 2.1-fold increase in the total numbers of LLO-specific CD4+ T cells (Figure 2.1B). Immunization with un-pulsed DCs or DCs pulsed with fMIG alone did not result in the generation of LLO-specific CD4+ T cells. Therefore, the observed increase in the total numbers of LLO-specific CD4+T cells correlated with the activation of fMIG-specific CD8+T cells since immunization with LLO alone generated fewer LLO-specific CD4+ T cells. As expected, recognition of the formylated peptide in the context of H2-M3 molecules on DCs induced robust proliferation and expansion of fMIG-specific CD8+ T cells, as determined by measuring  86 both their frequency and total numbers following restimulation of splenocytes from mice immunized with either fMIG or LLO + fMIG (Figure 2.1C and 2.1D). 2.3.2 CD4+ T cells generated with H2-M3-restricted T cell help improves protective immunity To determine whether Ag-specific CD4+ T cells generated with H2-M3-restricted T cell help was associated with enhanced protective immunity, we infected previously immunized mice with wildtype (wt) LM 6 days following immunization in order to boost pre-primed Ag-specific CD4+ T cell populations. We found that control and fMIG immunized mice, which lack pre-primed LLO-specific CD4+T cells, did not generate a measurable LLO-specific response 3 days following infection (Figure 2.2A). Interestingly, mice previously immunized with LLO + fMIG contained approximately 9.5fold higher numbers of Ag-specific CD4+ T cells, relative to Ag-specific CD4+ T cells generated in mice without H2-M3-restricted T cell help (Figure 2.2A). As shown in figure 2.2B, priming with fMIG in the absence or presence of LLO both led to significant numbers of fMIG-specific CD8 T cells. In order to establish whether an increase in the total number of LM-specific CD4+ T cells is associated with improved protection, the bacterial load in the spleens from these mice were quantified. We found that mice immunized with LLO demonstrate a 0.65 log reduction in bacterial load relative to control and fMIG immunized mice. Remarkably, mice immunized with both LLO and fMIG demonstrated a 1.3 log reduction in bacterial loads, relative to control and fMIG immunized mice (Figure 2.2C). The greater efficacy of fMIG + LLO immunized mice to eliminate LM may be due to either activated fMIG-specific CD8+ T cells and/or LLOspecific CD4 T cells. To distinguish between the relative contributions of these two cell  87 types in bacterial clearance, mice immunized with DCs coated with fMIG or LLO + fMIG were depleted of CD4+ T cells 3 days prior to infection, and on the day of infection with wt LM. In this experiment, the presence of fMIG-specific CD8+ T cells lead to a reduction of bacterial colonies in the spleen when compared to control mice, although this reduction is not statistically significant (P<0.1). Consistent with our previous results, LLO + fMIG immunized mice treated with PBS alone resulted in a significant 2.4- and 1.3-log reduction in the levels of bacteria in their spleens 3 days following infection, relative to control and fMIG immunized mice, respectively (Figure 2.2D). Importantly, the additional protection observed in LLO + fMIG immunized mice was abrogated by treatment with the GK1.5 mAb, which eliminates CD4+ T cells in vivo (Figure 2.2D). This result indicates that the reduction in bacterial load in fMIG + LLO immunized mice is likely due to augmented numbers of LLO-specific CD4+ T cells, generated with the help of H2-M3-restricted CD8+ T cells. We also found generating LLO-specific CD4+ T lymphocytes with H2-M3-restricted T cells activated on separated DCs, as opposed to the same DC, did not generate as many Ag-specific CD4+T cells (Figure 2.2E). This is observed even when twice the numbers of LLO-coated DCs were used for priming CD4+ T cells (Figure 2.2E). Thus, the observed increase in Ag-specific CD4+ T cells, generated with help from activated H2-M3-restricted T cells functioning in close proximity rendered these mice more resistant to LM infection. These results are in agreement with a recent study that demonstrates impaired immune response to LM infection in H2-M3-deficient mice (20).  88 2.3.3 Effective priming of Ag-specific CD4+ T cells generated with help from nonclassical H2-M3-restricted T cells augment conventional CD8+ T cell responses To rule out the possibility that conventional MHC class Ia-restricted CD8+T cells may function analogously to non-conventional H2-M3-restricted T cells in augmenting the number of Ag-specific CD4+T cells, we immunized mice with LLO-coated DCs ± fMIG peptide or GP33 peptide from LCMV. We found that mice immunized with DCs coated with either fMIG or GP33 peptides presented by MHC class Ib or MHC class Ia, respectively, lead to similar numbers of Ag-specific effector T cells 6 days following DC immunization (data not shown), which is consistent with previous studies that demonstrated quicker expansion of Ag-specific CD8 T cells occurred when using peptide-coated DC immunizations (21). These mice were subsequently infected 6 days later with a recombinant strain of LM that expresses GP33 (rLM-GP33) to boost the preprimed effector cells. The numbers of Ag-specific CD4+ and CD8+ T cells were analyzed 7 days later to study the ability of conventional CD8+ T cells to influence CD4+ T cell responses. As anticipated, the presence of fMIG-specific T cells was observed again only in mice immunized with LLO + fMIG (Figure 2.3A). In agreement with our previous observations, the priming of LLO-specific CD4+ T cells with H2-M3-restricted T cell help resulted in a significant 3.6-fold increase in the total number of LLO-specific CD4+ T cells, relative to the response generated in the absence of help from fMIG-specific CD8+ T cells (Figure 2.3B). More importantly, conventional MHC class Ia-restricted, GP33specific CD8+ T cells were unable to increase the numbers of LLO-specificCD4+ T lymphocytes (Figure 2.3B). Surprisingly, the expansion of LLO-specific CD4+ T cells appeared to be dampened when primed concurrently with conventional CD8+ T cells,  89 resulting in a 3-fold and a 12-fold decrease relative to LLO and LLO + fMIG immunizations, respectively. This observation is likely due to the killing of rLM-GP33infected antigen presenting cells by GP33-specific CD8+ T cells. Recent studies have demonstrated the ability for CD4+ T cells to undergo proliferation is contingent on the presence of antigen throughout their expansion phase (22). The presence of pre-primed CD8+ T cells, which possess cytolytic activity, may have led to the elimination of APCs that are required for activating Ag-specific CD4+ T cells. Consistent with this reasoning, mice receiving LLO + GP33 immunizations had a large population of GP33-specific CD8+ T cells following infection with rLM-GP33 (Figure 2.3C). These data further support the conclusion that the effector functions of MHC class Ib-restricted T cells are intrinsically different than MHC class Ia-restricted T cells. Similar results were obtained when GP33 was substituted with a second MHC class Ia-restricted peptide, SIY (SIYRYYGL) followed by infection with rLM-SIY (Figure 2.4), indicating the weaker generation of CD4+ T cell responses is not a unique property of GP33 and is likely representative of all MHC class Ia-restricted peptides. These studies also yielded a surprising finding. We found mice immunized with either LLO + fMIG or LLO + GP33 possessed a significant 3.3- and 3.4-fold higher number of GP33-specific CD8+ T cells, respectively, compared to mice immunized with LLO alone (Figure 2.3C). It is likely LLO-specific CD4+ T cells, whose number is in turn augmented as a result of co-priming with fMIG, mediate augmentation of the GP33specific CD8+ T cell response. We also found that mice immunized with GP33-coated DCs in the absence or presence of fMIG had similar numbers of GP33-specific CD8+ T cells 7 days following infection with rLM-GP33 (Figure 2.3D). This result is in  90 agreement with a previous study that found fMIG-specific H2-M3-restricted CD8+ T cells are unable to directly augment classical CD8+ T cell responses (23). 2.3.4 Enhancement of memory CD4+ and CD8+ T cell responses by H2-M3restricted T cells Following the effector phase of an immune response, the majority of Ag-specific CD4+ T cells undergo apoptosis with only a small population surviving, which constitutes longlived memory T cells (24). To determine if H2-M3-restricted T cells are able to produce quantitatively and qualitatively enhanced Ag-specific CD4+ memory T cells, mice were immunized with LLO-coated DCs in the presence or absence of fMIG peptide. Following infection of mice with rLM-GP33, Ag-specific CD4+ T cells were enumerated 30 days later to determine if H2-M3-restricted T cells help generate a larger pool of memory CD4+ T cells. As shown in figure 2.5A, there was a 4-fold increase in the absolute numbers of LLO-specific CD4+ T cells generated with H2-M3-restricted T cell help, relative to mice immunized without help from activated fMIG-specific CD8+ T cells. Furthermore, Ag-specific CD4+T cells generated with H2-M3-restricted T cell help produced approximately 20% more IFN-γ on a per-cell-basis, as determined by evaluating the geometric mean fluorescence intensity (GMFI) of antibody against IFN-γ (Figure 2.5B). Our findings thus indicate that H2-M3-restricted T cells support the enhancement of Ag-specific CD4+ T cells both quantitatively and qualitatively with respect to their increased persistence during the memory phase following infection, as well as their ability to produce higher amounts of IFN-γ, respectively. In agreement with our earlier observation that co-priming with LLO + fMIG can lead to enhanced primary GP33-specific CD8 T cell responses, we found that mice previously immunized with  91 LLO + fMIG also possess a larger population of memory GP33-specific CD8+ T cells (Figure 2.5C). Impressively, there was not a significant difference in the number of Agspecific CD8+ T cells between LLO + fMIG and GP33 + fMIG immunized mice, which represented a primary and secondary GP33-specific CTL response, respectively. Similar expansion of GP33-specific CD8 T cells was observed by immunizing mice with either DC-GP33, DC-(GP33 + LLO) or DC-(GP33 + fMIG) (data not shown). Our data thus indicate that the qualitatively and quantitatively enhanced Ag-specific CD4+ T cells associated with fMIG co-priming can lead to a vigorous primary response by GP33specific CD8+ T cells. 2.3.5 Qa-1-restricted CD8+ T cells enhance Ag-specific CD4+ T cell responses Since humans do not possess an ortholog of H2-M3 and human CD8+ T cells do not recognize fMIG, we wanted to establish whether another murine MHC class Ib molecule with a human equivalent could perform similarly in augmenting CD4+ T cell responses. In this regard, the mouse Qa-1 and human HLA-E molecules are functional counterparts, based on their ability to bind class I leader sequence-derived peptides and serve as a ligand for the CD94/NKG2A receptor complex (25, 26). Both Qa-1 and HLAE are able to bind and present the peptide GroEL (GMQFDRGYL), which is an immunodominant epitope of Salmonella typhimurium (7, 27). Recent work suggests that Qa-1-resticted CD8+ T cells can suppress the response of successfully activated CD4+ T cells through an interaction that depends on expression of Qa-1 molecules on the helper T cell (28-31). Since it has been established Qa-1-restricted CD8+ T cells can regulate ongoing CD4+ T cell responses, we wanted to determine whether Qa-1restricted CD8+ T cells can also influence the priming of Ag-specific CD4+T cells (28).  92 To address this, mice were immunized with DCs coated with LLO ± GroEL, and analyzed 6 days later. The priming of LLO-specific CD4+T cells with help from Qa-1restricted T cells led to a significant 3-fold increase, relative to mice that received just LLO-coated DCs alone (Figure 2.6A). In agreement with this observation, day 6 immune mice receiving LLO-coated DC immunizations in the presence of GroEL also contained approximately 2-fold higher numbers of Ag-specific CD4+ T cells, relative to DC-LLO immunizations alone 7 days following infection with wt LM (Figure 2.6B). Since LM does not express GroEL, this could account for the modest increase in the number of LLOspecific CD4+ T cells after LM infection in LLO + GroEL immunized mice. Because Qa-1 performs similar functions as H2-M3, these observations also explain why mice can mount effective immune responses to a mutant LM strain that is unable to add formyl groups to nascent polypeptides (32). Furthermore, these results suggest that Qa-1restricted CD8+ T cells may be involved in immune regulation on many levels, depending on when they are utilized during an immune response.  2.4 Discussion In this study, we have demonstrated a novel function for H2-M3-restricted T cells in enhancing Ag-specific CD4+ T cell responses following immunization, as well as during the acute, effector and memory phase following LM infection. During bacterial infection, H2-M3-restricted T cells are able to rapidly exert their effector functions upon receptor engagement by reaching peak frequencies earlier than their classical CD8+ T cell counterparts. This early response enables them to provide early protection to the  93 infected host. In this study, we have determined some of the basic parameters by which H2-M3-restricted CD8+ T cells participate in the priming of Ag-specific CD4+ T cells. We have demonstrated the necessity of activating H2-M3-restricted T cells on the same, as opposed to separate DC, for enhancing the priming of Ag-specific CD4+ T cells, suggesting cytokines functioning in a short and paracrine manner may be mediating this effect. Consistent with this notion, recent work by other groups have also demonstrated early production of cytokines, such as IFN-γ by natural killer (NK) cells, were able to influence DCs during the early phase of innate immunity, which can impact the quality and magnitude of the subsequent adaptive immune response (33, 34). Furthermore, others have also provided evidence that demonstrated memory-phenotype CD8+ T cells are also major contributors of early IFN-γ, even more so than NK cells during LM infection (34, 35). In agreement with these observations, preliminary data from our lab suggests cytokines produced by activated H2-M3-restricted CD8+ T cells are likely involved in mediating augmented Ag-specific CD4+ T cell responses, presumably by influencing DC maturation. These results support the hypothesis H2-M3-restricted T cells, and possibly other non-conventional MHC class Ib-restricted CD8+ T cells, including those restricted by Qa-1, function as immunoregulatory cells that have a significant influence on adaptive immunity. We have also demonstrated increased numbers of Ag-specific CD4+ T cells were able to confer a greater ability to eliminate LM from the spleens of infected mice. Since LM replicates primarily within macrophages (36), the augmented numbers of IFN-γproducing Ag-specific CD4+ T cells may be amplifying the host response by activating resident and newly recruited macrophages, or other innate cells including neutrophils  94 and NK cells, to become more bactericidal. Helper CD4+ T cells perform these functions by secreting cytokines, such as IFN-γ, and by up-regulating CD40L, both of which positively influence macrophages to become more efficient at removing harmful pathogenic organisms (37, 38). The importance of IFN-γ to host defense against LM infection has also been demonstrated in a number of previous studies showing increased susceptibility in mice with disrupted genes for IFN-γ (39) or lack the IFN- γ receptor (40), resulting in both impaired innate cell activity, failure to recruit other innate effector cells and the inability of macrophage cells to produce antimicrobial products despite the presence of normal cytotoxic CD8+ and CD4+ TH cell responses. Furthermore, in agreement with work done by other labs (23), mice immunized with DCs coated with fMIG were also able to modestly reduce bacterial levels in the spleens of infected immune mice, although this decrease was found not to be statistically significant 3 days following infection. The great degree of variability observed in both the splenic bacterial burden and magnitude of the fMIG-specific T cell response generated between genetically identical mice may be due to differential environmental factors that shaped their H2-M3-restricted T cell repertoire (21, 41). Helper CD4+ T lymphocytes also exert other pleiotropic effects including the ability to crosslink CD40 receptors on DCs. The engagement of CD40 on DCs leads to enhanced survival, increased expression of costimulatory molecules and increased secretion of inflammatory cytokines, all of which provide favorable conditions for stimulating CD8 T cells (37). Interestingly, our work has also demonstrated higher numbers of Ag-specific CD4+ T cells resulted in a subsequent increase in conventional Ag-specific CD8+ T cell numbers, which was approximately equivalent to the quantity  95 found in mice immunized with DCs coated with the respective MHC class Ia-specific peptide, during both the effector and memory phase following an immune response to LM infection. One explanation could be increased numbers of Ag-specific CD4+ T cells expressing CD40L could more efficiently license DCs to become more efficient APCs by either inducing the upregulation of B7 molecules or their ability to produce IL-12, both of which has been reported to dramatically improve CTL induction (37). Alternatively, increased frequency of CD40L expressed by Ag-specific CD4+ T cells could also induce the upregulation of 4-1BBL on DCs, which can induce both CD8+ T cell activation and survival (42). Consistent with this, 4-1BBL-deficient mice have reduced effector and memory CD8+ T cells following infection with LCMV (43, 44). These results suggest H2M3-restricted T cells, although unable to directly augment numbers of classical Agspecific CD8+ T cells, are able to do so indirectly. The functions of H2-M3-restricted T cells have long been enigmatic with respect to their specific contributions during immune responses. In this study, we have demonstrated a novel function for these cells in augmenting the numbers of Ag-specific CD4+ and CD8+ T cells, which could provide a novel method for enhancing weak immune responses to syngeneic tumors, since they are more dependent on CD4+ T cell help. More importantly, having identified a similar function for Qa-1, whose human equivalent is HLA-E, this study emphasizes the significance of targeting this and other non-polymorphic MHC class Ib molecules for future vaccine design strategies, which will help induce optimal CD4+and CD8+ T cell responses and thus, protective immunity.  96 2.5 Acknowledgments We thank Soo-Jeet Teh for general technical assistance and John Priatel and Edward Kim for helpful discussion.  97 Figures  Figure 2.1  Concurrent activation of H2-M3-restricted T cells enhances antigen-  specific CD4+ T cell responses. Mice were immunized intravenously with 1 x 106 DCs loaded with the corresponding peptides. Six days later, splenocytes from the immunized mice were stimulated with the indicated peptide(s) and the stimulated cells were stained for intracellular IFN- (A) The numbers indicate the percentage of LLO-specific CD4+ T cells in the spleens of mice following immunization with the indicated peptide(s). The  98 ovals in these dot plots indicate the gates for determining CD4+IFN-+ cells. (B) Total numbers of LLO-specific CD4+T cells in the spleens of mice following immunization with the indicated peptide(s). *, P<0.004 and **, P<0.004, compared with control. ***, P< 0.02, compared with LLO immunization. (C) The numbers indicate the percentage of fMIG-specific CD8+ T cells in the spleens of mice following immunization with the indicated peptide(s). The ovals in these dot plots indicate the gates for determining CD8+IFN-γ+ cells. (D) Total numbers of fMIG-specific CD8+ T cells in the spleens of mice following immunization with the indicated peptide(s). *, P<0.003 and **, P<0.05, compared with Control. Bars in (B) and (D) indicate means ± s.d. of 5 mice per group and are representative of 4 independent experiments.  99  Figure 2.2  Improved protective immunity when antigen-specific CD4+T cells  receive H2-M3-restricted T cell help on the same dendritic cell. Mice were immunized, as described in figure 2.1, and infected with 5 x 104 CFU wildtype LM 6 days later (A - C). The number of Ag-specific CD4+ and CD8+ T cells in the spleen was determined 3 days post-infection, as described in figure 2.1. (A) Total numbers of LLO-  100 specific CD4+ T cells in the spleens of mice immunized with the indicated peptide(s). *, P<0.001 and **, P<0.00008, compared with Control. ***, P<0.001, compared with LLO immunization. (B) Total numbers of fMIG-specific CD8+ T cells in the spleens of mice immunized with the indicated peptide(s). *, P<0.00002 and **, P<0.0004, compared with control. (C) The number of Listeria in the spleens of mice immunized with the indicated peptide(s) was determined. *, P<0.004 and **, P<0.0002, compared with control. ***, P<0.04, compared with LLO immunization. Bars in (A) – (C) indicate means ± s.d. of 5 – 6 mice per group. (D) The number of Listeria CFUs in the spleens from mice immunized with either no peptide, fMIG, or LLO + fMIG and treated either with PBS (filled bars) or GK1.5 in PBS (open bars) were determined. *, P<0.04, compared with control (PBS). **, P<0.03, compared with fMIG immunization (PBS) ***, P<0.0002 compared with LLO + fMIG immunization (GK1.5). Bars indicate means ± s.d. of 4 mice per group. (E) Mice were immunized with 1 x 106 DCs coated with LLO + fMIG peptides either on the same (unfilled) or on different (unfilled) DCs, using a mixture of 5 x 105 LLO + 5 x 105 fMIG (dark grey), 1 x 106 LLO + 1 x 106 fMIG (light grey), respectively. The total numbers of LLO-specific CD4+ T cells in the spleens of these mice 3 days following infection are displayed. *, P<0.002 and **, P<0.01, compared with immunization with LLO + fMIG coated on different DCs, dark and light grey, respectively. Bars indicate means ± s.d. of 5 mice per group and are representative of 3 independent experiments.  101  Figure 2.3  Antigen-specific CD4+T cells generated with help from non-classical  H2-M3-restricted T cells augment MHC class Ia-restricted CD8+ T cell responses. (A-C) Mice were immunized with 1 x 106 DCs coated with either LLO, LLO + fMIG, or LLO + GP33. (D) Mice were immunized with GP33 or GP33 + fMIG. At day 6 post immunization, all mice were infected with 5 x 104 CFUs of rLM-GP33. The numbers of Ag-specific CD4+ and CD8+ T cells in the spleens of infected mice were determined 7 days later, as described in figure 2.1. (A) Total numbers of fMIG-specific CD8+ T cells in the spleens of mice immunized with the indicated peptide(s). (B) Total numbers of LLOspecific CD4+T cells in the spleens of mice immunized with the indicated peptide(s). *, P<0.01, compared with LLO immunization. **, P<0.004, compared with LLO + GP33  102 immunization. (C) Total numbers of GP33-specific CD8+T cells in the spleens of mice immunized with the indicated peptide(s). *, P<0.01 and **, P<0.006,, compared with LLO immunization. (D) Total numbers of GP33-specific CD8+ T cells in the spleens of mice immunized with the indicated peptide(s). Error bars in figures indicate means ± s.d. of 3 (A-C) or 4 (D) mice per group.  103  Figure 2.4  Conventional CD8+ T cells cannot enhance LLO-specific CD4+ T cell  responses. Mice were immunized with 1 x 106 DCs coated with LLO peptide alone or in conjunction with fMIG or SIY peptide, corresponding to MHC class Ib- and MHC class Ia-restricted T cells, respectively. At day 6 post immunization, all mice were infected with 3 x 105 CFU of rLM-SIY. The numbers of Ag-specific CD4+ T cells in the spleens of infected immune mice were determined 7 days later, as described in figure 2.1. (A) Total numbers of LLO-specific CD4+T cells in the spleens of mice immunized with the indicated peptide(s). *, P<0.02, compared with LLO. **, P<0.002, compared with LLO + SIY immunization. Error bars in figures indicate means ± s.d. of 4 mice per group.  104  Figure 2.5  H2-M3-restricted T cells enhance the numbers of Ag-specific  CD4+and CD8+ memory T cells. Mice were immunized with 1 x 106 DCs coated with the indicated peptides and infected with 5 x 104 CFU rLM-GP33 6 days following  105 immunization. The numbers of Ag-specific CD4+and CD8+ memory T cells in the spleens of immunized mice was determined 30 days later, as described in figure 2.1. (A) Total numbers of LLO-specific CD4+T cells in the spleens of mice immunized with the indicated peptide(s). *, P<0.006, compared with LLO immunization. (B) A semiquantitative analysis of IFN- produced at the single-cell level determined for LLOspecific CD4+T cells from figure 2.5A. *, P<0.04, compared with LLO immunization. Bars represent the geometric mean fluorescence intensity (MFI) of antibody against IFN- in LLO-specific CD4+ T cells. (C) Total numbers of GP33-specific CD8+T cells in the spleen. *, P<0.001 and **, P<0.0007, compared with LLO immunization. Error bars in figures indicate means ± s.d. of 3 mice per group and are representative of 2 independent experiments.  106  Figure 2.6  Qa-1-restricted CD8+ T cells enhance Ag-specific CD4+T cell  responses. Mice were immunized with 1 x 106 DCs coated with the indicated peptides. Intracellular cytokine staining for IFN- was done on peptide-stimulated splenocytes to determine total numbers of LLO-specific CD4+ T cells six days following immunization (A), or seven days post rLM-gp33 infection of day 6 immune mice (B). (A) Total numbers of LLO-specific CD4+ T cells in the spleens of mice immunized with the indicated peptide(s). *, P<0.04 and **, P<0.005, compared with control. ***, P<0.04, compared with LLO. (B) Total numbers of LLO-specific CD4+ T cells in the spleens of infected immune mice. *, P<0.02, compared with LLO. Error bars in both (A) and (B)  107 indicate mean ± s.d. of 4 mice per group and are representative of 2 independent experiments.  108 2.6 References  1.  Banchereau, J., and R. M. Steinman. 1998. Dendritic cells and the control of immunity. Nature 392:245-252.  2.  Macatonia, S. E., N. A. Hosken, M. Litton, P. Vieira, C. S. Hsieh, J. A. Culpepper, M. Wysocka, G. Trinchieri, K. M. Murphy, and A. O'Garra. 1995. Dendritic cells produce IL-12 and direct the development of Th1 cells from naive CD4+ T cells. J Immunol 154:5071-5079.  3.  Koch, F., U. Stanzl, P. Jennewein, K. Janke, C. Heufler, E. Kampgen, N. Romani, and G. Schuler. 1996. High level IL-12 production by murine dendritic cells: upregulation via MHC class II and CD40 molecules and downregulation by IL-4 and IL-10. J Exp Med 184:741-746.  4.  Rissoan, M. C., V. Soumelis, N. Kadowaki, G. Grouard, F. Briere, R. de Waal Malefyt, and Y. J. Liu. 1999. Reciprocal control of T helper cell and dendritic cell differentiation. Science 283:1183-1186.  5.  De Smedt, T., M. Van Mechelen, G. De Becker, J. Urbain, O. Leo, and M. Moser. 1997. Effect of interleukin-10 on dendritic cell maturation and function. Eur J Immunol 27:1229-1235.  6.  Medzhitov, R. 2001. Toll-like receptors and innate immunity. Nat Rev Immunol 1:135-145.  109 7.  Lo, W. F., A. S. Woods, A. DeCloux, R. J. Cotter, E. S. Metcalf, and M. J. Soloski. 2000. Molecular mimicry mediated by MHC class Ib molecules after infection with gram-negative pathogens. Nat Med 6:215-218.  8.  Baron, J. L., L. Gardiner, S. Nishimura, K. Shinkai, R. Locksley, and D. Ganem. 2002. Activation of a nonclassical NKT cell subset in a transgenic mouse model of hepatitis B virus infection. Immunity 16:583-594.  9.  Beckman, E. M., S. A. Porcelli, C. T. Morita, S. M. Behar, S. T. Furlong, and M. B. Brenner. 1994. Recognition of a lipid antigen by CD1-restricted alpha beta+ T cells. Nature 372:691-694.  10.  Lenz, L. L., B. Dere, and M. J. Bevan. 1996. Identification of an H2-M3-restricted Listeria epitope: implications for antigen presentation by M3. Immunity 5:63-72.  11.  Kerksiek, K. M., D. H. Busch, I. M. Pilip, S. E. Allen, and E. G. Pamer. 1999. H2M3-restricted T cells in bacterial infection: rapid primary but diminished memory responses. J Exp Med 190:195-204.  12.  Urdahl, K. B., J. C. Sun, and M. J. Bevan. 2002. Positive selection of MHC class Ib-restricted CD8(+) T cells on hematopoietic cells. Nat Immunol 3:772-779.  13.  D'Orazio, S. E., D. G. Halme, H. L. Ploegh, and M. N. Starnbach. 2003. Class Ia MHC-deficient BALB/c mice generate CD8+ T cell-mediated protective immunity against Listeria monocytogenes infection. J Immunol 171:291-298.  110 14.  Seaman, M. S., B. Perarnau, K. F. Lindahl, F. A. Lemonnier, and J. Forman. 1999. Response to Listeria monocytogenes in mice lacking MHC class Ia molecules. J Immunol 162:5429-5436.  15.  Seaman, M. S., C. R. Wang, and J. Forman. 2000. MHC class Ib-restricted CTL provide protection against primary and secondary Listeria monocytogenes infection. J Immunol 165:5192-5201.  16.  Shedlock, D. J., J. K. Whitmire, J. Tan, A. S. MacDonald, R. Ahmed, and H. Shen. 2003. Role of CD4 T cell help and costimulation in CD8 T cell responses during Listeria monocytogenes infection. J Immunol 170:2053-2063.  17.  Lutz, M. B., N. Kukutsch, A. L. Ogilvie, S. Rossner, F. Koch, N. Romani, and G. Schuler. 1999. An advanced culture method for generating large quantities of highly pure dendritic cells from mouse bone marrow. J Immunol Methods 223:7792.  18.  Hamilton, S. E., and J. T. Harty. 2002. Quantitation of CD8+ T cell expansion, memory, and protective immunity after immunization with peptide-coated dendritic cells. J Immunol 169:4936-4944.  19.  Ploss, A., G. Lauvau, B. Contos, K. M. Kerksiek, P. D. Guirnalda, I. Leiner, L. L. Lenz, M. J. Bevan, and E. G. Pamer. 2003. Promiscuity of MHC class Ibrestricted T cell responses. J Immunol 171:5948-5955.  111 20.  Xu, H., T. Chun, H. J. Choi, B. Wang, and C. R. Wang. 2006. Impaired response to Listeria in H2-M3-deficient mice reveals a nonredundant role of MHC class Ibspecific T cells in host defense. J Exp Med 203:449-459.  21.  Ploss, A., I. Leiner, and E. G. Pamer. 2005. Distinct regulation of H2-M3restricted memory T cell responses in lymph node and spleen. J Immunol 175:5998-6005.  22.  Obst, R., H. M. van Santen, D. Mathis, and C. Benoist. 2005. Antigen persistence is required throughout the expansion phase of a CD4(+) T cell response. J Exp Med 201:1555-1565.  23.  Hamilton, S. E., B. B. Porter, K. A. Messingham, V. P. Badovinac, and J. T. Harty. 2004. MHC class Ia-restricted memory T cells inhibit expansion of a nonprotective MHC class Ib (H2-M3)-restricted memory response. Nat Immunol 5:159-168.  24.  Homann, D., L. Teyton, and M. B. Oldstone. 2001. Differential regulation of antiviral T-cell immunity results in stable CD8+ but declining CD4+ T-cell memory. Nat Med 7:913-919.  25.  Braud, V. M., D. S. Allan, C. A. O'Callaghan, K. Soderstrom, A. D'Andrea, G. S. Ogg, S. Lazetic, N. T. Young, J. I. Bell, J. H. Phillips, L. L. Lanier, and A. J. McMichael. 1998. HLA-E binds to natural killer cell receptors CD94/NKG2A, B and C. Nature 391:795-799.  112 26.  Lee, N., D. R. Goodlett, A. Ishitani, H. Marquardt, and D. E. Geraghty. 1998. HLA-E surface expression depends on binding of TAP-dependent peptides derived from certain HLA class I signal sequences. J Immunol 160:4951-4960.  27.  Lo, W. F., H. Ong, E. S. Metcalf, and M. J. Soloski. 1999. T cell responses to Gram-negative intracellular bacterial pathogens: a role for CD8+ T cells in immunity to Salmonella infection and the involvement of MHC class Ib molecules. J Immunol 162:5398-5406.  28.  Jiang, H., H. Kashleva, L. X. Xu, J. Forman, L. Flaherty, B. Pernis, N. S. Braunstein, and L. Chess. 1998. T cell vaccination induces T cell receptor Vbetaspecific Qa-1-restricted regulatory CD8(+) T cells. Proc Natl Acad Sci U S A 95:4533-4537.  29.  Noble, A., Z. S. Zhao, and H. Cantor. 1998. Suppression of immune responses by CD8 cells. II. Qa-1 on activated B cells stimulates CD8 cell suppression of T helper 2 responses. J Immunol 160:566-571.  30.  Jiang, H., and L. Chess. 2000. The specific regulation of immune responses by CD8+ T cells restricted by the MHC class Ib molecule, Qa-1. Annu Rev Immunol 18:185-216.  31.  Hu, D., K. Ikizawa, L. Lu, M. E. Sanchirico, M. L. Shinohara, and H. Cantor. 2004. Analysis of regulatory CD8 T cells in Qa-1-deficient mice. Nat Immunol 5:516-523.  113 32.  D'Orazio, S. E., C. A. Shaw, and M. N. Starnbach. 2006. H2-M3-restricted CD8+ T cells are not required for MHC class Ib-restricted immunity against Listeria monocytogenes. J Exp Med 203:383-391.  33.  Martin-Fontecha, A., L. L. Thomsen, S. Brett, C. Gerard, M. Lipp, A. Lanzavecchia, and F. Sallusto. 2004. Induced recruitment of NK cells to lymph nodes provides IFN-gamma for T(H)1 priming. Nat Immunol 5:1260-1265.  34.  Vitale, M., M. Della Chiesa, S. Carlomagno, D. Pende, M. Arico, L. Moretta, and A. Moretta. 2005. NK-dependent DC maturation is mediated by TNFalpha and IFNgamma released upon engagement of the NKp30 triggering receptor. Blood 106:566-571.  35.  Dhanji, S., M. T. Chow, and H. S. Teh. 2005. Self-Antigen Maintains the Innate Anti-Bacterial Function of Self-Specific CD8 T cells in Vivo. J Immunol.  36.  Guleria, I., and J. W. Pollard. 2001. Aberrant macrophage and neutrophil population dynamics and impaired Th1 response to Listeria monocytogenes in colony-stimulating factor 1-deficient mice. Infect Immun 69:1795-1807.  37.  Cella, M., D. Scheidegger, K. Palmer-Lehmann, P. Lane, A. Lanzavecchia, and G. Alber. 1996. Ligation of CD40 on dendritic cells triggers production of high levels of interleukin-12 and enhances T cell stimulatory capacity: T-T help via APC activation. J Exp Med 184:747-752.  38.  Boehm, U., T. Klamp, M. Groot, and J. C. Howard. 1997. Cellular responses to interferon-gamma. Annu Rev Immunol 15:749-795.  114 39.  Harty, J. T., and M. J. Bevan. 1995. Specific immunity to Listeria monocytogenes in the absence of IFN gamma. Immunity 3:109-117.  40.  Huang, S., W. Hendriks, A. Althage, S. Hemmi, H. Bluethmann, R. Kamijo, J. Vilcek, R. M. Zinkernagel, and M. Aguet. 1993. Immune response in mice that lack the interferon-gamma receptor. Science 259:1742-1745.  41.  Kerksiek, K. M., D. H. Busch, and E. G. Pamer. 2001. Variable immunodominance hierarchies for H2-M3-restricted N-formyl peptides following bacterial infection. J Immunol 166:1132-1140.  42.  Takahashi, C., R. S. Mittler, and A. T. Vella. 1999. Cutting edge: 4-1BB is a bona fide CD8 T cell survival signal. J Immunol 162:5037-5040.  43.  DeBenedette, M. A., T. Wen, M. F. Bachmann, P. S. Ohashi, B. H. Barber, K. L. Stocking, J. J. Peschon, and T. H. Watts. 1999. Analysis of 4-1BB ligand (41BBL)-deficient mice and of mice lacking both 4-1BBL and CD28 reveals a role for 4-1BBL in skin allograft rejection and in the cytotoxic T cell response to influenza virus. J Immunol 163:4833-4841.  44.  Tan, J. T., J. K. Whitmire, R. Ahmed, T. C. Pearson, and C. P. Larsen. 1999. 41BB ligand, a member of the TNF family, is important for the generation of antiviral CD8 T cell responses. J Immunol 163:4859-4868.  115 Chapter 3  H2-M3-restricted CD8+ T cells augment CD4+ T cell responses by promoting dendritic cell maturation 1  3.1 Introduction Unlike conventional CD8+ T cells, nonconventional T cells, including γδ T cells (1), NKT cells (2), and H2-M3-restricted T cells (3) possess properties that are shared by cells of both the innate and adaptive immune systems. They express T cell receptors (TCRs) with limited antigen (Ag) diversity suggesting that they recognize conserved structures rather than the diverse set of Ags recognized by lymphocytes of the adaptive immune system (4). Additionally, they exhibit a memory-like phenotype rather than a naïve phenotype, which may explain their rapid response characterized by the production of cytokines with innate-like kinetics (5). Since DCs can acquire a mature phenotype by integrating signals derived from early innate sources, DCs exposed to early sources of cytokines present in the milieu, or cell-cell contacts delivered by innate and/or innatelike cells, likely have the potential to influence the quality and magnitude of downstream adaptive immune responses.  Evidence from both human and murine studies demonstrated the ability of natural killer (NK) cells to influence adaptive immune responses by enhancing DC function. Early sources of IFN-γ produced by NK cells have been shown to polarize CD4+ T cell 1  A version of this chapter has been accepted for publication. Chow, M.T. and Teh, H. S. (2010)  H2-M3-restricted CD8+ T cells augment CD4+ T cell responses by promoting dendritic cell maturation.  116 responses towards the TH1 lineage (4, 6, 7). Interestingly, recent work has identified some CD8+ T cell subsets to be an even greater source of innate cytokines (8, 9). Specifically, the activation of some MHC class Ib-restricted T cells leads to rapid TNF-α and IFN-γ production, in vivo (4, 10). Moreover, other studies have shown that some of these “innate-like” lymphocytes are involved in immune regulation (4, 11, 12). Interestingly, during infection with Listeria monocytogenes (LM), H2-M3-restricted T cells become activated and reached peak frequencies sooner than conventional MHC class Ia-restricted T cells (13, 14). Although a source of N-formylated peptides of host origin can be found in the mitochondria, a function for H2-M3-restricted T cells responding to mitochondrially-derived formylated peptides has not yet been identified (15). However, with respect to immune regulation, we showed that H2-M3-restricted T cells are able to participate in Ag-specific CD4+ T cell priming, in vivo (16). When Agspecific CD4+ T cells were primed concurrently with H2-M3-restricted T cells, the ensuing CD4+ T cell response was augmented following immunization, as well as during the effector and memory phases following LM infection (16). In this report, we showed that peptide-stimulated H2-M3-restricted T cells can induce the maturation of DCs. The ability of activated H2-M3-restricted T cells to rapidly expand and produce proinflammatory cytokines that induce DC maturation exemplifies how these cells are uniquely positioned to augment the impending adaptive CD4+ T cell response. These findings could lead to novel strategies for the generation of more effective vaccines.  117 3.2 Materials and methods  3.2.1 Mice Breeders for C57BL/6 (B6), B6.Thy1.1 and OT-II mice were obtained from The Jackson Laboratories. OT-II TCR transgenic mice express a TCR that is specific for chicken ovalbumin amino acid residues 323-339 presented by I-Ab (41). Mice 6 – 12 weeks of age were used for the experiments described. All animal procedures were conducted in accordance with the Canadian Council on Animal Care guidelines. 3.2.2 Antibodies and peptides The following mAbs specific for the indicated molecule were used: CD8α (53-6.7), CD8β (53.58), CD3ε (145-2C11), NK1.1 (PK136), IFN-γ (XMG1.2), TNF-α (MP6-XT22), IL-2 (JES6-5H4), IL-4 (BVD6-24G2), IL-12p40 (17.8) , I-Ab (M5/114.15.2), CD40 (HM40-3), Thy1.2 (53-2.1), CD80 (16-10A1), CD86 (GL1), TCRβ (H57-597), CD11c (N418), ICAM1 (YN1/1.7.4), Annexin V, and Ki-67 (B56). All mAbs were purchased from eBioscience, except for Ki-67 and Annexin V-PE, which was purchased from BD Pharmingen. The detection of intracellular cytokines by flow cytometry was conducted as described by Hamilton et al. (42). Staining for Ki-67 antigen and Annexin V was conducted as described by Priatel et al. (43). The CellQuest program (BD Biosciences, San. José, CA ) was used for data acquisition and analysis. Data was acquired using either a FACScan or FACSCalibur (BD Biosciences). The ovalbumin peptide OVA323-339 was synthesized at Sigma-Genosys (Oakville, Ontario). The fMIGWII (fMIG) peptide was synthesized at Bio-Synthesis (Lewisville, Texas).  118 3.2.3 Generation of bone marrow-derived dendritic cells (BMDCs) BMDCs were generated by 8 days of culture as described by Lutz et al (44). The nonadherent cell population consisted of >95% CD11c+ cells following positive selection using the MiniMACS system (Miltenyi Biotec) according to the manufacturer’s specifications. DCs were incubated with the indicated peptide dose for 3 hours at 37°C and washed extensively with PBS before serving as APCs in CD4+ T cell proliferation assays. 3.2.4 Generation and purification of H2-M3-restricted CD8+ T cells B6 mice were immunized via tail vein injection using DC-fMIG (1 x 106 cells). The frequency and total numbers of fMIG-specific CD8+ T cells was determined by intracellular cytokine staining for IFN- as describted by Hamilton et al. (42). The splenocytes were treated with mAbs against CD4, CD11c, CD45R, CD49b, Ter-119 and NK1.1, followed by depletion of Ab-coated cells using Dynabeads M-450 sheep antimouse IgG (Dynal Biotech), according to manufacturer’s instructions. The enriched population of cells is typically >90% CD8+. 3.2.5 Generation and purification of IL-2 activated NK cells Splenocytes of B6 mice were treated with mAbs against CD4, CD8, and TCRβ, followed by depletion of Ab-coated cells using Dynabeads M-450 sheep anti-mouse IgG (Dynal Biotech), according to manufacturer’s instructions. The resulting cells were approximately 50-60% CD3-NK1.1+ before culture and >95% CD3-NK1.1+ after 4 day culture in IL-2 (50U/mL).  119 3.2.6 In vitro co-culture maturation assay iDCs (5 x 105) were plated in 96-well flat-bottom plates with 2 x 103 H2-M3-restricted T cells (fMIG-primed H2-M3-restricted T cell: DC ratio = 1:250) and analyzed for maturation markers following 1 or 2 days of coculture. In cytokine neutralization experiments, saturating amounts of cytokine blocking antibodies (5µg/mL) were added at the onset of T cell:DC coculture. 3.2.7 Adoptive transfer experiments fMIG peptide-stimulated H2-M3-restricted T cells were cocultured with iDCs to induce DC maturation 24 hours prior to performing a proliferation assay. These conditioned DCs were purified 24 hours after the coculture, pulsed with titrated concentrations of OVA323-339 peptides, washed with PBS, fixed with 1% paraformaldehyde to prevent any further maturation, and then placed into 96-flat well tissue culture plates containing OTII CD4+ T cells. Tritiated thymidine was added to the cultures during the last 6 hours of incubation of a 72 hr incubation period and incorporation of thymidine was measured using liquid scintillation counting. 3.2.8 Adoptive-transfer experiments Thy1.2+ OT-II cells were purified from the spleens and lymph nodes of naïve donors by incubating the single-cell suspension with CD8α, CD45R, CD11b and NK1.1 for 45 minutes on ice, followed by negative selection using sheep anti-mouse IgG Dynabeads (Miltenyi Biotec) according to manufacturer’s protocol. The resulting cells were consistently >85% CD4+ and Vα2+. OT-II T cells (1 x 107/ml) were labeled with 1 µM CFSE (Molecular Probes, Eugene, OR) in PBS for 8.5 min at room temperature. After  120 stopping the reaction with the addition of an equal volume of FCS, cells were washed four times with complete media to eliminate excess dye. 3.2.9 Statistical analysis Data for Figure 3.2 were analyzed by ANOVA, with Dunnett’s post-hoc tests where DCs cocultured with non-peptide-stimulated fMIG-primed CD8+ T cells was set as the reference. All other data were analyzed by Student t test. Significance was set at pvalues of < 0.05. 3.3 Results  3.3.1 Simultaneous activation of H2-M3-restricted CD8+ T cells enhances the proliferation of transgenic CD4+ T cells We recently reported that H2-M3-restricted CD8+ T cells participate in the in vivo priming of Ag-specific CD4+ T cells, a property not shared by conventional CD8+ T cells that are restricted by MHC class Ia molecules[29]. Although we demonstrated that H2M3-restricted T cells help prime endogenous antigen-specific CD4+ T cell responses, we wanted to confirm if similar results are achieved using adoptively transferred transgenic CD4+ T cells. To assess the potential influence of H2-M3-restricted T cells on the proliferation of transgenic OT-II CD4+ T cells (Thy-1.2), which recognizes OVA323-339 presented by I-Ab, OT-II cells were labeled with carboxyfluorescein diacetate succinimidyl diester (CFSE) and adoptively transferred into congenic Thy-1.1 C57BL/6 (B6) recipient mice [30]. These recipient mice were subsequently immunized with purified CD11c+ bone marrow-derived dendritic cells (DCs) coated with OVA323-339  121 peptide alone or in conjunction with the fMIGWII (fMIG) peptide, which is the dominant H2-M3-binding formylated peptide found in LM, 24 hours later to prime both the transferred OT-II T cells and endogenous fMIG-specific H2-M3-restricted T cells [31]. The frequencies of the transferred OT-II T cells were determined 3 days post immunization by staining splenocytes with chromophore-conjugated anti-Thy-1.2 and anti-CD4+ antibodies followed by flow cytometric analysis. In contrast to mice immunized with OVA323-339-coated DCs alone, which resulted in a modest but significant expansion of OT-II T cells, there was a 1.8-fold increase in the frequency of OT-II T cells in mice primed with DCs coated with both OVA and fMIG peptides (Figure 3.1A). This translated to approximately 1.9-fold increase in the total number of OT-II CD4+ T cells (Figure 3.1B). As expected, immunization of mice with DCs not coated with OVA peptide did not induce OT-II cell division (data not shown). This observation is consistent with our previous report demonstrating the ability of H2-M3-restricted T cells to augment endogenously generated antigen-specific CD4+ T cell responses [29]. Since adoptively transferred OT-II T cells were labeled with CFSE, we next measured whether the activation of H2-M3-restricted T cells induced enhanced cell division and/or expansion by analyzing CFSE fluorescence of the transferred Thy-1.2+CD4+ cells. The CFSE data in figure 3.1C indicate that the CD4+ T cells primed with OVA peptide-coated DCs either alone or in conjunction with H2-M3-restricted T cells both underwent the same number of division cycles. However, the concurrent activation of H2-M3-restricted T cells during priming of OT-II T cells lead to greater clonal expansion leading to increased numbers of OT-II cells (Figure 3.1C). This result demonstrates that  122 simultaneous activation of H2-M3-restricted T cells and OT-II CD4+ T cells on the same DC is required for more effective clonal expansion. 3.3.2 Activated H2-M3-restricted CD8+ T cells induce the maturation of dendritic cells Previous work done in our lab showed that the augmented Ag-specific CD4+ T cell response was dependent on the activation of H2-M3-restricted CD8+ T cells and Agspecific CD4+ T cells on the same DC [29]. The requirement for the activation of H2-M3restricted T cells and CD4+ T cells on the same DC suggests the possibility that activated H2-M3-restricted T cells may affect CD4+ T cells by influencing the maturation status of DCs. Since it is well established that DCs can prime strong effector T cell responses once they receive appropriate maturation signals, such as from infection, crosslinking of Toll-like receptors or CD40 ligation [32-36], we wanted to determine if the activation of H2-M3-restricted T cells can indeed induce DC maturation. Since the precursor frequency of H2-M3-restricted T cells are below the limit of detection in the steady state [7], mice were immunized with fMIG peptide coated DCs 6 days prior to cell coculture to expand the population of fMIG-specific H2-M3-restricted T cells. The frequency of fMIG-specific T cells in the spleen of primed mice was consistently around 2 – 4% of the CD8+ T cell population (data not shown). In order to obtain H2-M3restricted T cells free from magnetic beads or tetramers that can interfere with their functions, splenocytes taken from DC-fMIG immune mice were enriched for total CD8+ T cells using negative selection before they were cocultured with DCs. The DCs were cocultured with purified CD8+ T cells in the absence or presence of the fMIG peptide to induce DC maturation; LPS-activated DCs were used as internal controls for DC  123 maturation. To exclude the possibility DC maturation was due to endotoxin contamination of the fMIG peptide or binding of the fMIG peptide to N-formyl peptide receptors (fMLPRs) on phagocytes, BMDCs were also treated with 1μM and 5μM fMIG peptide. After a 24 and 48 hour incubation period, DCs cultured either alone or with nonstimulated H2-M3-restricted T cells did not lead to a significant increase in the percentage of gated CD11c+ cells expressing CD40, I-Ab (MHC class II), or CD80, relative to LPS treated DCs, respectively (Figure 3.2A & 3.2C). The same is true for DCs cultured with either naïve CD8+ T cells purified from non-peptide pulsed DC immunized mice or DCs cultured with just the fMIG peptide alone indicating that DC maturation observed in our experimental groups was not due to endotoxin contamination or stimulation via fMLPRs (data not shown).We found that DCs cocultured with stimulated H2-M3-restricted T cells for 24 hr contained significantly greater frequencies of CD40, CD80 and I-Ab expressing CD11c+ cells, which represented an average increase of approximately 72%, 16% and 37%, relative to DCs cocultured with nonstimulated (- fMIG) H2-M3-restricted T cells, respectively (Figure 3.2A). Similar increases were also observed 48 hours post coculture (Figure 3.2C). DCs cocultured for 24 hours with peptide-stimulated H2-M3-restricted T cells also had greater cell surface expression levels of CD40, I-Ab and CD80 (Figure 3.2B). Similar increases for cell surface DC maturation markers were also observed relative to the control groups 48 hours post culture (Figure 3.2D). Since the cell surface concentration of MHC class II molecules has been shown to influence the magnitude and the outcome of the CD4+ T cell response [37-39], it is significant that DCs cocultured with either activated fMIG-specific CD8+ T cells or LPS had significantly higher levels of MHC class  124 II on their cell surface, compared to samples in the control group. Interestingly, unlike DCs activated by LPS treatment, DCs cocultured with peptide-stimulated H2-M3restricted T cells maintained elevated levels of MHC class II molecules on the cell surface 48 hours post culture (Figure 3.2D). Therefore, the activation of a single subset of T cells, restricted by the MHC class Ib molecule,H2-M3, is sufficient to induce the upregulation of several cell surface maturation markers on DCs with kinetics that are distinct from LPS stimulation.  3.3.3 Both soluble factors and cell-to-cell contact contribute to the induction of DC maturation by H2-M3-restricted T cells The increased CD40 expression level on the surface of DCs is one of the key indicators that the DC has receive a maturation signal. In order to determine the relative importance of cell contact-dependent and independent mechanisms in the induction of DC maturation by H2-M3-restricted T cells, a porous transwell membrane system was used. For cell contact-independent interactions, fMIG-pulsed DCs were cocultured with pre-primed H2-M3-restricted T cells in the upper chamber, with the lower chamber containing only DCs. For cell contact-dependent interactions, unpulsed DCs were placed in the upper chamber to normalize for total cell numbers between experimental groups, with the lower chamber containing fMIG-pulsed DCs cocultured with pre-primed H2-M3-restricted T cells. In these experiments, the measurement of CD40 upregulation was conducted only for the experimental DCs that resided in the lower chambers of the experimental groups. As expected, DCs cultured in the absence of peptide-stimulated H2-M3-restricted T cells did not upregulate CD40, regardless of whether cell-to-cell  125 contact was permitted or not (Figure 3.3A and 3.3B). In agreement with the results in figure 3.2, there was a 2.8 -fold increase in the proportion of DCs that upregulated the expression of cell surface CD40 when the DCs were in direct contact with peptidestimulated H2-M3-restricted T cells (Figure 3.3A, upper panel).By contrast, the absence of direct cell-to-cell contact between peptide-stimulated H2-M3-restricted T cells and DC led to a smaller increase (1.9-fold) in CD40 expression level by the DCs (Figure 3.3A, lower panel). The percentage of DCs expressing high levels of CD40 is also greatest when DCs and activated H2-M3-restricted T cells were in direct contact (Figure 3.3A and 3.3B). These results illustrate the importance of cell contact-dependent molecular interactions in inducing the maturation of DCs. However, since there was a modest increase in both the upregulation and frequency of CD40-expressing DCs in the absence of DC and H2-M3-restricted T cell contact, we next determined whether activated H2-M3-restricted T cells can produce cytokines that induce DC maturation. Previous studies have shown that CD8+ T cells restricted to nonclassical MHC class Ib molecules have a memory phenotype and is a very significant source of early IFN-γ during infections, comparable to NK cells [15, 40, 41]. We found that activated H2-M3restricted T cells are major producers of IFN-γ and TNF-α (Figure 3.3C). To evaluate the contributions of these two cytokines in inducing DC maturation, DCs were cocultured together with stimulated or nonstimulated H2-M3-restricted T cells. The percent decrease in CD40 expression, resulting from neutralization of either IFN-γ or TNF-α or both cytokines during coculture, was measured. TNF-α was found to be more significant at inducing DC maturation, since neutralizing this cytokine alone led to a more significant reduction in CD40 geometric mean fluorescence intensity (GMFI) than  126 when only IFN-γ was neutralized, relative to isotype control (Figure 3.3D). Although the neutralization of IFN-γ alone did not result in significant CD40 downregulation when TNF-α was present, removal of IFN-γ simultaneously with TNF-α resulted in an additive reduction in CD40 GMFI suggesting that both cytokines contribute to DC maturation. Interestingly, in the absence of these two cytokines, the presence of cell-to-cell contact was insufficient to maintain elevated levels of CD40, which is also true for I-Ab and CD80 (data not shown). These data suggest that cell-to-cell contact and cytokines produced by H2-M3-restricted T cells are both required for inducing DC maturation. 3.3.4 H2-M3-restricted T cells are more efficient than NK cells at inducing DC maturation Several studies have identified cross talk between NK cells and Ag-presenting cells (APCs), resulting in DC maturation quantified by the upregulation of several costimulatory molecules and the generation of more effective T cell responses [42-45]. Maturation of APCs was strictly dependent on the use of IL-2activated NK cells presiding in low ratios during DC coculture [45]. To determine if fMIG-specific H2-M3restricted T cells were more effective at inducing DC maturation than NK cells, DCs were separately cocultured with the two different effector cell types (fMIG-specific H2M3-restricted CD8+ T cells and NK cells, respectively) that were serially diluted from the highest effector/target (E/T) ratio of 1:250 down to the lowest E/T ratio of 1:1000. After 24 hours, cell surface expression of CD40 was measured as an indication of DC maturation. DCs cocultured with peptide-stimulated H2-M3-restricted T cells achieved significantly higher levels of CD40 on a per cell basis at all E/T ratios used, relative to DCs cocultured with either non-peptide stimulated H2-M3-restricted T cells or with IL-2-  127 activated NK cells (Figure 3.4A). In agreement with previous studies, IL-2-activated NK cells cocultured with DCs at a higher 1:5 ratio significantly upregulated CD40, relative to NK cells cocultured with DCs at a 1:500 and 1:250 E:T ratio (Figure 3.4B) [46]. This result illustrates that activated H2-M3-restricted T cells are more efficient than IL-2activated NK cells in inducing DC maturation.  3.3.5 DCs co-cultured with activated H2-M3-restricted T cells produced higher levels of pro-inflammatory cytokines Although the upregulation of costimulatory molecules is one of the criteria used to measure DC maturation, the other criterion of a mature DC is the production of proinflammatory cytokines [47]. To ascertain if H2-M3-restricted T cells induce the full program of DC maturation, DCs were cocultured with titrated numbers of non-activated or peptide-activated H2-M3-restricted T cells. Since the culture contained cells other than DCs, cytokines were measured through the use of intracellular staining following a 5-hour treatment with a protein transport inhibitor. DCs cultured with peptide-stimulated H2-M3-restricted T cells had significantly higher frequencies of TNF-α- and IL-12p40producing DCs, relative to DCs cocultured with nonstimulated H2-M3-restricted T cells, even at a 1000:1 ratio (Figure 3.5A and 3.5B). A closer look at the cytokine profile at different ratios revealed that DCs cocultured with low ratios of peptide-activated H2-M3restricted T cells produced high levels of TNF-α, whereas the production of IL-12p40 was consistently high for all ratios of H2-M3-restricted T cells to DCs (Figure 3.5C and  128 3.5D). These data suggest that coculture of DC with stimulated H2-M3-restricted T cells is sufficient to induce the full program of DC maturation. 3.3.6 DCs matured by activated H2-M3-restricted T cells lower the activation threshold for OT-II T cells Since the ability to stimulate and expand naïve T cells for the generation of effective T cell immunity is dependent on fully matured DCs, we wanted to establish whether DCs matured by H2-M3-restricted T cells can help prime suboptimally activated CD4+ T cells. OT-II CD4+ T cells, which recognize OVA323-339presented by I-Ab, were placed into tissue culture wells containing serially diluted concentrations of plate bound anti-CD3 (2C11) monoclonal antibody to induce their activation across a broad spectrum of activation thresholds. DCs that were cocultured previously with or without peptideactivated H2-M3-restricted T cells for 24 hours were purified, pulsed with OVA323-339 peptide and subsequently fixed with 1% paraformaldehyde to prevent any further maturation. These APCs were then added to the wells to determine if they can lower the activation threshold of CD4+ T cells that received sub-optimal activation signals. After a 72 hour incubation, the proliferation of CD4+ T cells were assessed by measuring [H]3thymidine incorporation during the last 6 hours of incubation. The results indicate that fixed, OVA323-339-pulsed DCs previously cocultured with peptide-stimulated H2-M3restricted T cells were able to augment OT-II T cell responses, particularly in cultures with low 2C11 concentrations. By contrast, OVA323-339-pulsed DCs that were cocultured with unstimulated H2-M3-restricted T cells were inefficient in augmenting OT-II T cell responses that were primed with low concentrations of 2C11 (Figure 3.6). These findings suggest that activated H2-M3-restricted CD8+ T cells provide early innate  129 sources of cytokines and cell-cell interactions to induce DC maturation. The fully matured DCs in turn were able to augment immune responses by CD4+ T cells, particularly those that received a suboptimal Ag stimulus.  3.4 Discussion The activation and subsequent maturation of DCs is the primary event enabling the translation of innate signals into adaptive immunity. Maturation is a terminal differentiation process that transforms DCs from poor immunostimulatory cells specialized for Ag capture, into cells specialized for T cell stimulation. As previously reported, augmented CD4+ T cell responses was dependent on the concurrent activation of Ag-specific CD4+ T cells with nonconventional H2-M3-restricted T cells, a feature conventional CD8+ T cells cannot perform [29]. Additionally, enhanced CD4+ T cell response was dependent on having both DCs and nonconventional CD8+ T cells activated on the same, as opposed to separate peptide-pulsed DCs, alluding to the possibility H2-M3-restricted T cells may be influencing the function of DCs [29]. In this study, we extended our previous observations by functionally characterizing and confirming the ability of H2-M3-restricted T cells to induce the functional maturation of DCs. We have shown that these innate-like CD8+ T cells upregulate several costimulatory molecules on DCs via both cell-to-cell contact and cytokine-dependent mechanisms. Maturation was accompanied by increased production of TNF-α and IL-12 cytokines by DCs. We also demonstrate that peptide-stimulated H2-M3-restricted T cells were more efficient at inducing DC maturation, relative to IL-2-activated NK cells.  130 Finally, we found that DCs that interacted with activated H2-M3-restricted T cells were functionally mature, since they were able to augment CD4+ T cell responses that received sub-optimal activation signals. After infection with LM, H2-M3 primes the rapid expansion of CD8+ T cells by presenting N-formylated bacterial peptides. Their rapid activation and expansion, which occurs earlier than MHC class Ia-restricted T cells, may enable them to interact with immature DCs during the earliest time points of a bacterial infection [48, 49]. Although microbial products such as LPS are known to be effective at promoting DC maturation (Figure 3.2), we made the unusual observation that the cell surface expression levels of I-Ab molecules were downregulated 48 hours after in vitro LPS treatment, while DCs matured by activated H2-M3-restricted T cells remained high (Figure 3.2). It is possible that microbial stimuli alone may be downregulating I-Ab expression due to a lack of receiving any T cell-derived signals, which may lead to an “exhausted” DC population. In agreement with this notion, others have shown that in the absence of additional hostderived signals, LPS treatment promotes a short burst of IL-12 leading to an exhausted DC population [50]. The importance of host T cells on both the development and function of DCs has also been established in experiments in Rag-2-/- mice [51]. In these T cell-deficient mice, the DCs were inefficient in Ag presentation and the activation of naïve T cells. These results support the hypothesis that H2-M3-restricted T cells can provide sufficient signals to aid the development of efficient immune responses by providing favorable conditions for inducing DC maturation. We have also demonstrated that lower numbers of peptide-activated H2-M3restricted T cells were needed for inducing the maturation of DCs relative to IL-2  131 activated NK cells, which in recent years have been shown to be an efficient inducer of DC maturation [12, 52]. A simplistic explanation for this observation may be due to the ability of innate-like CD8+ T cells to produce greater levels of proinflammatory cytokines. This would account for why fewer H2-M3-resticted T cells were needed to induce DC maturation, relative to NK cells (Figure 3.3). In agreement with this notion, previous work in our lab have shown other innate-like CD8+ T cells that exhibit similar innate-like properties, such as rapid cytokine production, can produce approximately 2-fold more IFN-γ and 3-fold more TNF-, relative to NK cells [41]. Irrespective of the exact mechanism for why greater numbers of NK cells are needed to induce DC maturation, it is of interest to note that H2-M3-restricted CD8+ T cells are efficient in influencing downstream adaptive immune responses. In addition to H2-M3-restricted T cells and NK cells, NKT cells and γδ T cells have also been shown to be able to influence the maturation state of DCs [53, 54]. Since the expression patterns of MHC class Ib genes are generally more tissue-restricted, relative to the wide tissue distribution of MHC class Ia genes, these MHC class Ib-restricted CD8+ T cells may serve to ensure efficient T cell activation in specific anatomical locations. Furthermore, MHC class Ib-restricted CD8+ T cells could also synergize with microbial products or other innate-like cells such as NK cells in inducing DC maturation [52, 55]. In agreement with our previous report, H2-M3-restricted CD8+ T cells were able to enhance the in vivo responses of transplanted OVA-specific OT-II TCR-transgenic T cells (Figure 3.1). The ability for low numbers of fMIG-primed CD8+ T cells to induce DC proinflammatory cytokine production and upregulation of CD40 (Figure 3.3 and 3.4), along with their rapid expansion and effector functions during primary responses [8, 17,  132 40], may partly explain how the low in vivo precursor frequencies of these MHC class Ib-restricted T cells can positively influence CD4+ T cell responses without prior expansion. An additional factor that may contribute to augmented CD4+ T cell responses could be due to the ability of early signals delivered by H2-M3-restricted CD8+ T cells to trigger survival signals in DCs. In agreement with this explanation, previous studies have found the lifespan of Ag-bearing DCs is influenced by signals derived from pathogens and T cells [56]. Therefore, the ability for H2-M3-restricted T cells to positively influence DCs may be due to both enhanced DC lifespan and stable expression of peptide-MHC class II complexes on DC cell surface, which together would provide favorable conditions for optimal CD4+ T cell activation (Figure 3.1 and Figure 3.2). Collectively, these results support the hypothesis that H2-M3-restricted T cells can function as immunoregulatory cells that have significant influences on adaptive immunity [53, 57]. The observation that H2-M3-deficient mice exhibit a reduction in both bacterial clearance and innate effector functions after LM infection also attests to the importance of H2-M3-restricted T cells in defense against intracellular bacterial infections [26]. Further work with these mice are needed to determine whether in the course of LM infection, H2-M3-restricted T cells participate in the priming of Ag-specific CD4+ T cell responses against listerial epitopes, as well as in the generation of effective memory responses against LM infection. The presence of MHC class Ib-restricted T cells in humans raises the possibility of exploiting the limited polymorphism of their T cell receptors [58] and their ability to augment immune responses by conventional CD4+  133 T cells in the design of vaccines to generate more effective immune responses against microbial infections and cancer.  3.5 Acknowledgments We thank Soo-Jeet Teh for technical assistance and the Westbrook Animal Unit for animal husbandry. This research is supported by the Canadian Cancer Society (Grant # 019458 to HST). Michael T. Chow is supported by the Natural Sciences and Engineering Council of Canada  134 Figures  Figure 3.1  Activation of H2-M3-restricted T cells augments DC immunogenicity  in vivo. CFSE-labeled OT-II T cells (2 x 106 cells) purified from the spleens and lymph nodes of Thy1.2+ OT-II transgenic mice were adoptively transferred into Thy1.1+ C57BL/6 mice. Recipient mice were immunized 1 day later using 1 x 106 immature DCscoated with either OVA323-339alone (5µM) or with both OVA and fMIG peptides (1µM).  135 (A) Frequency of OT-II cells was determined by staining for CD4+Thy1.2+ cells. The value within the dot plots represents the frequency of OT-II cells within the oval gate. Results are shown in duplicate and are representative of 2 independent experiments using 3 mice per group. (B) Total numbers of OT-II cells in the spleen of mice immunized with DCs-coated with OVA alone (Black bars) or with both OVA and fMIG peptides (White bars). Error bars represent the mean  standard deviation of 6 mice per group. Data are representative of 2 independent experiments ( *, p < 0.04, compared with OVA). (C) Histogram illustrate the CFSE-division profile of OT-II cells from mice immunized with OVA-coated DCs alone (Left Histogram) or with DCs coated with both OVA and fMIG peptides (Right Histogram). Expansion of OT-II T cell populations was determined by counting of cells and assay of the dilution of CFSE. Numbers above bracketed lines indicate the number of average cell divisions in each event. Data are representative of 2 independent experiments.  136  Figure 3.2  Activation of H2-M3-restricted T cells induces dendritic cell  maturation. Unstimulated (- fMIG) or stimulated (+ fMIG) H2-M3-restricted CD8+ T cells were cocultured with immature dendritic cells (DCs) at a ratio of 1:250 corresponding to 2 x 103 H2-M3-restricted CD8+ T cells for every 5 x 105 DCs. Controls include DCs cultured either in medium alone, or medium containing LPS (5ng/mL). DCs were then analyzed by staining for cell surface costimulatory molecules CD40, I-Ab or CD80 after pre-incubating DCs with 2.4G2 mAb (specific for FcγII/IIIR) in order to minimize nonspecific binding. (A and C) Bar graphs indicate the percentage of costimulatory molecule positive cells on the surface of gated CD11c+ cells 24 hours and 48 hours post coculture after subtracting for non-specific staining with the respective isotype mAb controls, respectively. Data represent the mean of triplicate measurements and are  137 representative of three similar experiments with similar trends (A: *, p ≤ 0.0002, relative to unstimulated group, ANOVA/Dunnett’s. C: *, p ≤ 0.003, relative to unstimulated group, ANOVA/Dunnett’s. ) (B and D) Bar graphs indicate the geometric mean fluorescence intensity (GMFI) of total CD11c+ cells 24 and 48 hours post coculture after subtracting for non-specific staining with the respective isotype mAb controls, respectively. Data represent the mean of triplicate measurements and are representative of three experiments (B: *, p ≤ 0.004, compared to unstimulated group, ANOVA/Dunnett’s. D: *, p ≤ 0.0002, compared to unstimulated group, ANOVA/Dunnett’s.) All error bars represent the mean  standard deviations of triplicate measurements.  138  Figure 3.3  Induction of DC maturation by H2-M3-restricted T cells is dependent  on cell-cell contact and TNF-α. For cell contact-independent interactions (- Contact), the upper chamber of the transwell setup contained fMIG-pulsed DCs (2 x 106 cells) cocultured with pre-primed H2-M3-restricted T cells (2 x 103 cells), whereas the lower chamber consists only of DCs (2 x 106 cells). For cell contact-dependent interactions (+ Contact), the upper chamber of the transwell contained unpulsed DCs (2 x 106 cells), whereas the lower chamber consisted of fMIG-pulsed DCs (2 x 106 cells) cocultured with pre-primed H2-M3-restricted T cells (2 x 103 cells). DCs residing in the lower chamber of both groups were analyzed 24 hours later by flow cytometry for the expression of CD40. (A) The GMFI of CD40 is indicated (Filled histograms). Isotype control was included to identify background levels (Unfilled histograms). Horizontal bars represent markers assigned on the basis of including all CD40+ cells above background  139 levels enabling the quantification and comparison of the average cell surface CD40 expression after different treatments. Results are representative of 3 independent experiments. (B) The percentage of CD11c+ cells positive for CD40 expression is indicated. Data represent the mean of triplicate measurements and are representative of 2 independent experiments (*, p < 0.002, **, p < 0.0003, relative to nonactivated fMIG-primed CD8+ T cells). Error bars represent the mean  standard deviations of triplicate measurements. (C) Restimulation of splenocytes taken from mice immunized with fMIG-coated DCs 6 days earlier was done to confirm the cytokines made by activated fMIG-specific H2-M3-restricted T cells. Values indicate the frequency of IFNγ+CD8+ and TNF-α+CD8+ cells after restimulation with the fMIG peptide. (D) DCs were cultured for 24 hours in the presence of non-activated or activated H2-M3-restricted T cells, along with either isotype mAb (Solid Line) or neutralizing mAb against either IFNγ, TNF-α, or both (Shaded). Isotype control for fluorescent mAb was also performed to determine background for staining (Dotted Unfilled). Numbers indicate the percent downregulation of CD40 GMFI [(GMFI Isotype – GMFI Neutralizing mAb)/GMFI Isotype X 100], relative to DCs treated to isotype mAb. Data represent the mean of triplicate measurements and are representative of two experiments (*, p < 0.00004, relative to mAb isotype control treatment). Error bars represent the mean  standard deviations of triplicate measurements.  140  Figure 3.4  H2-M3-restricted T cells are more efficient than NK cells at inducing  DC maturation. Immature DCs (5 x 105 cells) were cocultured with effector cells consisting of either IL-2 activated NK cells, non-activated or fMIG-peptide activated H2M3-restricted T cells at the indicated effector: DC ratios. (A) Line graph indicates the GMFI of CD40 on the cell surface of DCs cultured with either NK cells (- -▲- -), nonactivated H2-M3-restricted T cells (—■—) or activated H2-M3-restricted T cells (—▲—). Error bars represent standard deviations of the mean of triplicate measurements. (B) Histogram indicates the GMFI of CD40 on the surface of DCs 24 hours after coculture  141 with purified IL-2 activated NK cells at the NK:DC ratio of 1:500 (Grey Unfilled), 1:250 (Black Unfilled), and 1:5 (Grey Filled). Background levels are shown by staining DCs with isotype mAb (Dotted Unfilled). Numbers indicate the GMFI of CD40 on the surface of CD11c+ cells. The horizontal bar represents markers assigned on the basis of including all gated CD11c+ cells. Numbers indicate the GMFI of CD40 on the surface of CD11c+ cells within the horizontal bar gate. Results are representative of 2 independent experiments with similar results.  142  Figure 3.5  Increased pro-inflammatory cytokine production by DCs when co-  cultured with activated H2-M3-restricted T cells. Immature DCs (5 x 105 cells) were cocultured for 24 hours with non-activated or fMIG-peptide activated H2-M3-restricted T cells at the indicated H2-M3-restricted T cell:DC ratios. Intracellular cytokine transport was blocked by adding brefeldin A (1mg/mL) to the tissue culture wells for the last 5 hours of coculture. The value shown in the dot plots represents the percentage of CD11c+ cells producing (A) TNF-α or (C) IL-12p40. Results are representative of 2 independent experiments. Line graphs indicate the percentage of DCs producing (B) TNF-α or (D) IL-12p40 after coculture with either non-activated (—□—) or activated H2M3-restricted T cells (—■—). Error bars represent standard deviations of the mean of triplicate measurements. Results are representative of 2 independent experiments with similar results.  143  Figure 3.6  DCs that have interacted with activated H2-M3-restricted T cells are  functionally mature and can enhance CD4+ T cell responses, in vitro. Activated H2M3-restricted T cells (2 x 103 cells) were cocultured with DCs (5 x 105 cells) to induce DC maturation. Immature DCs that were cocultured with non-activated H2-M3-restricted T cells were used as controls. These conditioned DCs were purified 24 hours after the coculture, fixed with 1% paraformaldehyde to prevent any further maturation, and the fixed DCs (~2 x 105 cells) were placed into 96-flat well tissue culture plates containing OT-II CD4+ T cells (5 x 104 cells) and serially titrated concentrations of plate-bound 2C11 at the indicated concentrations. Tritiated thymidine was added to the cultures during the last 6 hours of incubation of a 72 hr incubation period. Tritiated thymidine incorporation was measured by liquid scintillation counting. Error bars represent standard deviations of the mean of triplicate measurements. Results are representative of 2 independent experiments with similar results.  144 3.6 References  1.  Spada, F. M., E. P. Grant, P. J. Peters, M. Sugita, A. Melian, D. S. Leslie, H. K. Lee, E. van Donselaar, D. A. Hanson, A. M. Krensky, O. Majdic, S. A. Porcelli, C. T. Morita, and M. B. Brenner. 2000. Self-recognition of CD1 by gamma/delta T cells: implications for innate immunity. The Journal of experimental medicine 191:937-948.  2.  Li, H., M. I. Lebedeva, A. S. Llera, B. A. Fields, M. B. Brenner, and R. A. Mariuzza. 1998. Structure of the Vdelta domain of a human gammadelta T-cell antigen receptor. Nature 391:502-506.  3.  Eberl, G., R. Lees, S. T. Smiley, M. Taniguchi, M. J. Grusby, and H. R. MacDonald. 1999. Tissue-specific segregation of CD1d-dependent and CD1dindependent NK T cells. J Immunol 162:6410-6419.  4.  Arase, H., N. Arase, K. Ogasawara, R. A. Good, and K. Onoe. 1992. An NK1.1+ CD4+8- single-positive thymocyte subpopulation that expresses a highly skewed T-cell antigen receptor V beta family. Proceedings of the National Academy of Sciences of the United States of America 89:6506-6510.  5.  Wang, C. R., A. R. Castano, P. A. Peterson, C. Slaughter, K. F. Lindahl, and J. Deisenhofer. 1995. Nonclassical binding of formylated peptide in crystal structure of the MHC class Ib molecule H2-M3. Cell 82:655-664.  6.  Bendelac, A., M. Bonneville, and J. F. Kearney. 2001. Autoreactivity by design: innate B and T lymphocytes. Nat Rev Immunol 1:177-186.  145 7.  Kerksiek, K. M., D. H. Busch, I. M. Pilip, S. E. Allen, and E. G. Pamer. 1999. H2M3-restricted T cells in bacterial infection: rapid primary but diminished memory responses. The Journal of experimental medicine 190:195-204.  8.  Su, J., R. E. Berg, S. Murray, and J. Forman. 2005. Thymus-dependent memory phenotype CD8 T cells in naive B6.H-2Kb-/-Db-/- animals mediate an antigenspecific response against Listeria monocytogenes. J Immunol 175:6450-6457.  9.  Martin-Fontecha, A., L. L. Thomsen, S. Brett, C. Gerard, M. Lipp, A. Lanzavecchia, and F. Sallusto. 2004. Induced recruitment of NK cells to lymph nodes provides IFN-gamma for T(H)1 priming. Nat Immunol 5:1260-1265.  10.  Steimle, V., C. A. Siegrist, A. Mottet, B. Lisowska-Grospierre, and B. Mach. 1994. Regulation of MHC class II expression by interferon-gamma mediated by the transactivator gene CIITA. Science (New York, N.Y 265:106-109.  11.  Fernandez-Botran, R., V. M. Sanders, T. R. Mosmann, and E. S. Vitetta. 1988. Lymphokine-mediated regulation of the proliferative response of clones of T helper 1 and T helper 2 cells. The Journal of experimental medicine 168:543558.  12.  Moretta, A. 2002. Natural killer cells and dendritic cells: rendezvous in abused tissues. Nat Rev Immunol 2:957-964.  13.  Berg, R. E., C. J. Cordes, and J. Forman. 2002. Contribution of CD8+ T cells to innate immunity: IFN-gamma secretion induced by IL-12 and IL-18. Eur J Immunol 32:2807-2816.  146 14.  Berg, R. E., E. Crossley, S. Murray, and J. Forman. 2003. Memory CD8+ T cells provide innate immune protection against Listeria monocytogenes in the absence of cognate antigen. The Journal of experimental medicine 198:1583-1593.  15.  Berg, R. E., E. Crossley, S. Murray, and J. Forman. 2005. Relative contributions of NK and CD8 T cells to IFN-gamma mediated innate immune protection against Listeria monocytogenes. J Immunol 175:1751-1757.  16.  Bregenholt, S., P. Berche, F. Brombacher, and J. P. Di Santo. 2001. Conventional alpha beta T cells are sufficient for innate and adaptive immunity against enteric Listeria monocytogenes. J Immunol 166:1871-1876.  17.  Urdahl, K. B., J. C. Sun, and M. J. Bevan. 2002. Positive selection of MHC class Ib-restricted CD8(+) T cells on hematopoietic cells. Nature immunology 3:772779.  18.  Born, W. K., C. L. Reardon, and R. L. O'Brien. 2006. The function of gammadelta T cells in innate immunity. Current opinion in immunology 18:31-38.  19.  Sharp, L. L., J. M. Jameson, D. A. Witherden, H. K. Komori, and W. L. Havran. 2005. Dendritic epidermal T-cell activation. Critical reviews in immunology 25:118.  20.  Ikehara, Y., Y. Yasunami, S. Kodama, T. Maki, M. Nakano, T. Nakayama, M. Taniguchi, and S. Ikeda. 2000. CD4(+) Valpha14 natural killer T cells are essential for acceptance of rat islet xenografts in mice. The Journal of clinical investigation 105:1761-1767.  21.  Seino, K. I., K. Fukao, K. Muramoto, K. Yanagisawa, Y. Takada, S. Kakuta, Y. Iwakura, L. Van Kaer, K. Takeda, T. Nakayama, M. Taniguchi, H. Bashuda, H.  147 Yagita, and K. Okumura. 2001. Requirement for natural killer T (NKT) cells in the induction of allograft tolerance. Proceedings of the National Academy of Sciences of the United States of America 98:2577-2581. 22.  Smyth, M. J., K. Y. Thia, S. E. Street, E. Cretney, J. A. Trapani, M. Taniguchi, T. Kawano, S. B. Pelikan, N. Y. Crowe, and D. I. Godfrey. 2000. Differential tumor surveillance by natural killer (NK) and NKT cells. The Journal of experimental medicine 191:661-668.  23.  Terabe, M., S. Matsui, N. Noben-Trauth, H. Chen, C. Watson, D. D. Donaldson, D. P. Carbone, W. E. Paul, and J. A. Berzofsky. 2000. NKT cell-mediated repression of tumor immunosurveillance by IL-13 and the IL-4R-STAT6 pathway. Nature immunology 1:515-520.  24.  Lenz, L. L., B. Dere, and M. J. Bevan. 1996. Identification of an H2-M3-restricted Listeria epitope: implications for antigen presentation by M3. Immunity 5:63-72.  25.  Seaman, M. S., C. R. Wang, and J. Forman. 2000. MHC class Ib-restricted CTL provide protection against primary and secondary Listeria monocytogenes infection. J Immunol 165:5192-5201.  26.  Xu, H., T. Chun, H. J. Choi, B. Wang, and C. R. Wang. 2006. Impaired response to Listeria in H2-M3-deficient mice reveals a nonredundant role of MHC class Ibspecific T cells in host defense. The Journal of experimental medicine 203:449459.  27.  Shawar, S. M., J. M. Vyas, J. R. Rodgers, and R. R. Rich. 1994. Antigen presentation by major histocompatibility complex class I-B molecules. Annual review of immunology 12:839-880.  148 28.  Lindahl, K. F., D. E. Byers, V. M. Dabhi, R. Hovik, E. P. Jones, G. P. Smith, C. R. Wang, H. Xiao, and M. Yoshino. 1997. H2-M3, a full-service class Ib histocompatibility antigen. Annu Rev Immunol 15:851-879.  29.  Chow, M. T., S. Dhanji, J. Cross, P. Johnson, and H. S. Teh. 2006. H2-M3restricted T cells participate in the priming of antigen-specific CD4+ T cells. J Immunol 177:5098-5104.  30.  Robertson, J. M., P. E. Jensen, and B. D. Evavold. 2000. DO11.10 and OT-II T cells recognize a C-terminal ovalbumin 323-339 epitope. J Immunol 164:47064712.  31.  Ploss, A., G. Lauvau, B. Contos, K. M. Kerksiek, P. D. Guirnalda, I. Leiner, L. L. Lenz, M. J. Bevan, and E. G. Pamer. 2003. Promiscuity of MHC class Ibrestricted T cell responses. J Immunol 171:5948-5955.  32.  Lanzavecchia, A., and F. Sallusto. 2001. Regulation of T cell immunity by dendritic cells. Cell 106:263-266.  33.  Shakhar, G., R. L. Lindquist, D. Skokos, D. Dudziak, J. H. Huang, M. C. Nussenzweig, and M. L. Dustin. 2005. Stable T cell-dendritic cell interactions precede the development of both tolerance and immunity in vivo. Nat Immunol 6:707-714.  34.  Guermonprez, P., J. Valladeau, L. Zitvogel, C. Thery, and S. Amigorena. 2002. Antigen presentation and T cell stimulation by dendritic cells. Annu Rev Immunol 20:621-667.  35.  Mellman, I., and R. M. Steinman. 2001. Dendritic cells: specialized and regulated antigen processing machines. Cell 106:255-258.  149 36.  Bonifaz, L. C., D. P. Bonnyay, A. Charalambous, D. I. Darguste, S. Fujii, H. Soares, M. K. Brimnes, B. Moltedo, T. M. Moran, and R. M. Steinman. 2004. In vivo targeting of antigens to maturing dendritic cells via the DEC-205 receptor improves T cell vaccination. The Journal of experimental medicine 199:815-824.  37.  Fukui, Y., T. Ishimoto, M. Utsuyama, T. Gyotoku, T. Koga, K. Nakao, K. Hirokawa, M. Katsuki, and T. Sasazuki. 1997. Positive and negative CD4+ thymocyte selection by a single MHC class II/peptide ligand affected by its expression level in the thymus. Immunity 6:401-410.  38.  Hanson, M. S., M. Cetkovic-Cvrlje, V. K. Ramiya, M. A. Atkinson, N. K. Maclaren, B. Singh, J. F. Elliott, D. V. Serreze, and E. H. Leiter. 1996. Quantitative thresholds of MHC class II I-E expressed on hemopoietically derived antigenpresenting cells in transgenic NOD/Lt mice determine level of diabetes resistance and indicate mechanism of protection. J Immunol 157:1279-1287.  39.  Otten, L. A., V. Steimle, S. Bontron, and B. Mach. 1998. Quantitative control of MHC class II expression by the transactivator CIITA. Eur J Immunol 28:473-478.  40.  Kurepa, Z., J. Su, and J. Forman. 2003. Memory phenotype of CD8+ T cells in MHC class Ia-deficient mice. J Immunol 170:5414-5420.  41.  Dhanji, S., M. T. Chow, and H. S. Teh. 2006. Self-antigen maintains the innate antibacterial function of self-specific CD8 T cells in vivo. J Immunol 177:138-146.  42.  Raulet, D. H. 2004. Interplay of natural killer cells and their receptors with the adaptive immune response. Nature immunology 5:996-1002.  43.  Fernandez, N. C., A. Lozier, C. Flament, P. Ricciardi-Castagnoli, D. Bellet, M. Suter, M. Perricaudet, T. Tursz, E. Maraskovsky, and L. Zitvogel. 1999. Dendritic  150 cells directly trigger NK cell functions: cross-talk relevant in innate anti-tumor immune responses in vivo. Nature medicine 5:405-411. 44.  Gerosa, F., B. Baldani-Guerra, C. Nisii, V. Marchesini, G. Carra, and G. Trinchieri. 2002. Reciprocal activating interaction between natural killer cells and dendritic cells. The Journal of experimental medicine 195:327-333.  45.  Piccioli, D., S. Sbrana, E. Melandri, and N. M. Valiante. 2002. Contact-dependent stimulation and inhibition of dendritic cells by natural killer cells. The Journal of experimental medicine 195:335-341.  46.  Semino, C., J. Ceccarelli, L. V. Lotti, M. R. Torrisi, G. Angelini, and A. Rubartelli. 2007. The maturation potential of NK cell clones toward autologous dendritic cells correlates with HMGB1 secretion. Journal of leukocyte biology 81:92-99.  47.  Sporri, R., and C. Reis e Sousa. 2005. Inflammatory mediators are insufficient for full dendritic cell activation and promote expansion of CD4+ T cell populations lacking helper function. Nature immunology 6:163-170.  48.  Mercado, R., S. Vijh, S. E. Allen, K. Kerksiek, I. M. Pilip, and E. G. Pamer. 2000. Early programming of T cell populations responding to bacterial infection. J Immunol 165:6833-6839.  49.  Wong, P., and E. G. Pamer. 2001. Cutting edge: antigen-independent CD8 T cell proliferation. J Immunol 166:5864-5868.  50.  Langenkamp, A., M. Messi, A. Lanzavecchia, and F. Sallusto. 2000. Kinetics of dendritic cell activation: impact on priming of TH1, TH2 and nonpolarized T cells. Nat Immunol 1:311-316.  151 51.  Shreedhar, V., A. M. Moodycliffe, S. E. Ullrich, C. Bucana, M. L. Kripke, and L. Flores-Romo. 1999. Dendritic cells require T cells for functional maturation in vivo. Immunity 11:625-636.  52.  Cooper, M. A., T. A. Fehniger, A. Fuchs, M. Colonna, and M. A. Caligiuri. 2004. NK cell and DC interactions. Trends Immunol 25:47-52.  53.  Leslie, D. S., M. S. Vincent, F. M. Spada, H. Das, M. Sugita, C. T. Morita, and M. B. Brenner. 2002. CD1-mediated gamma/delta T cell maturation of dendritic cells. The Journal of experimental medicine 196:1575-1584.  54.  Munz, C., R. M. Steinman, and S. Fujii. 2005. Dendritic cell maturation by innate lymphocytes: coordinated stimulation of innate and adaptive immunity. The Journal of experimental medicine 202:203-207.  55.  Singer, D. S., and J. E. Maguire. 1990. Regulation of the expression of class I MHC genes. Critical reviews in immunology 10:235-257.  56.  Hou, W. S., and L. Van Parijs. 2004. A Bcl-2-dependent molecular timer regulates the lifespan and immunogenicity of dendritic cells. Nature immunology 5:583-589.  57.  Yu, K. O., and S. A. Porcelli. 2005. The diverse functions of CD1d-restricted NKT cells and their potential for immunotherapy. Immunology letters 100:42-55.  58.  O'Callaghan, C. A., and J. I. Bell. 1998. Structure and function of the human MHC class Ib molecules HLA-E, HLA-F and HLA-G. Immunological reviews 163:129-138.  59.  Barnden, M. J., J. Allison, W. R. Heath, and F. R. Carbone. 1998. Defective TCR expression in transgenic mice constructed using cDNA-based alpha- and beta-  152 chain genes under the control of heterologous regulatory elements. Immunology and cell biology 76:34-40. 60.  Lutz, M. B., N. Kukutsch, A. L. Ogilvie, S. Rossner, F. Koch, N. Romani, and G. Schuler. 1999. An advanced culture method for generating large quantities of highly pure dendritic cells from mouse bone marrow. Journal of immunological methods 223:77-92.  61.  Stockinger, B., T. Zal, A. Zal, and D. Gray. 1996. B cells solicit their own help from T cells. The Journal of experimental medicine 183:891-899.  62.  Hamilton, S. E., and J. T. Harty. 2002. Quantitation of CD8+ T cell expansion, memory, and protective immunity after immunization with peptide-coated dendritic cells. J Immunol 169:4936-4944.  153 Chapter 4  General discussion and perspectives  4.1 Adjuvant-like properties of MHC class Ib-restricted T cells This thesis has focused on the unique ability of a subset of MHC class Ib-restricted CD8+ T cells to provide early innate signals resulting in their ability to influence the impending adaptive immune response. Specifically, chapter 2 reported on the basic parameters required for H2-M3-restricted CD8+ T cells to participate in the priming of Ag-specific CD4+ T cells, in vivo (1). The concurrent activation of Ag-specific H2-M3restricted T cells generated augmented CD4+ T cell responses when they were primed using the same DCs, resulting in an increase in bacterial clearance, which correlated with an increase in protective immunity. The ability of H2-M3-restricted T cells to enhance CD4+ T cell numbers occurred during the acute, effector and memory phase of the immune response following bacterial infection, and is a property that is not shared by conventional MHC class Ia-restricted T cells, since the latter are unable to perform similar adjuvant-like functions. The most exciting part of this chapter was the discovery that MHC class Ib-restricted T cells that recognize antigens presented by Qa-1b, the murine functional homologue of human HLA-E, possess similar abilities to augment CD4+ T cell responses. Thus, the findings in chapter 2 are not limited to mice and may extend to relevance within the human immune system. Chapter 3 went on to show that the activation of Ag-primed H2-M3-restricted T cells leads to the functional maturation of DCs, characterized by costimulatory molecule upregulation and proinflammatory cytokine production. Maturation of DCs that is induced by H2-M3-restricted T cells is mediated by both cell-to-cell contact and cytokines. This chapter also demonstrated that  154 activated H2-M3-restricted T cells are more efficient at inducing DC maturation than activated NK cells. The results in this chapter provided a mechanistic explanation for how the concurrent activation of CD4+ and H2-M3-restricted T cells on the same DC resulted in augmented CD4+ T cell responses. The main findings presented in this thesis have been based on results obtained using peptide-coated DC immunizations. This system was used since it provided the ability to study and analyze the consequence of multiple cell-to-cell interactions in a relatively physiological environment. Since H2-M3-restricted T cells produce early sources of type 1 cytokines such as TNF-α and IFN-γ, which can influence downstream immune responses, the work in chapter two was aimed at elucidating the potential for H2-M3-restricted T cells to participate in the induction of CD4+ TH1 cell responses. Specifically, this chapter demonstrates how utilizing Ag-specific H2-M3-restricted T cells during the priming of Ag-specific CD4+ T cells leads to the production of a more potent CD4+ T cell response. More importantly, the augmented CD4+ T cell response also leads to the generation of greater Ag-specific CTL numbers. This observation highlights the pleiotropic effects of augmented CD4+ T cell responses on multiple aspects of cellmediated immunity. However, the studies in chapter 3 were limited by the inability to obtain a pure source of Ag-specific H2-M3-restricted T cells as a result of the low frequency of these cells in normal mice. Despite this limitation, the work in this chapter established the proof of principle of the ability of pre-primed Ag-specific H2-M3restricted T cells to augment CD4+ T cell responses by inducing the maturation of DCs. In summary, in addition to providing rapid effector functions to protect mice against some forms of bacterial and viral infections, H2-M3-restricted T cells, and presumably T  155 cells restricted by other MHC class Ib molecules, are able to provide early cues to DCs, which lead to enhancement of antigen-specific CD4+ T cell responses. 4.1.1 The relationship between conventional antigen-experienced memory CD8+ T cells and nonconventional innate-like CD8+ T cells A key event during lymphocyte development is the generation of a T cell repertoire that is purged of autoreactive cells through clonal deletion in the thymus (2). Since autoreactive T cells express TCRs that possess high affinity for self-antigens, the mature T cells that exit the thymus are expected to express TCRs with relatively low affinity for self-antigens. As a result, most mature CD8+ T cells that exit the thymus exhibit a naïve phenotype that is characterized by low expression of the memory cell surface marker CD44 (CD44low). Upon encountering their cognate antigen on a mature DC, these naïve CD8+ T cells undergo clonal expansion and differentiate into effector cells, a process that takes approximately three to five days (3). After reaching peak frequencies on days six to eight, and participating in pathogen clearance by killing infected cells and producing cytokines, 90-95% of the expanded effector cells undergo apoptosis leaving a small population of conventional Ag-experienced memory CD8+ T cells. These memory cells can persist for several years and possess the capacity of responding with greater vigor upon re-encountering the same pathogen, relative to naïve antigen-inexperienced CD8+ T cell (4-6). Despite the long standing belief that the development of conventional naïve CD8+ T cells into memory T cells involved this single binary decision process, it is now apparent that CD4+CD8+ thymocytes can also give rise to many lineages of mature T cells with unique characteristics. Specifically, in contrast to conventional CD8+ T cells, several other subsets of mature nonconventional  156 T cells that develop in the thymus display an activated and/or memory T cell phenotype as a result of their maturation process, rather than as a consequence of the activation, proliferation and differentiation that occur after antigen encounter in the periphery. Furthermore, in stark contrast to naïve conventional CD8+ T cells, these nonconventional CD8+ T cells with a “memory” phenotype possess innate-like characteristics that are associated with their ability to constitutively express transcription factors that regulate effector genes and the subsequent immune response (7-9). One of the hallmark characteristics of innate-like CD8+ T cells, which include cells with TCRs specific for nonclassical MHC class Ib molecules including H2-M3 and Qa-1b, is their ability to be positively selected in the thymus by interactions with MHC molecules expressed on hematopoietic cells, rather than on thymic epithelial cells (1014). Presumably as a result of their relatively high affinity for self-antigens and selection by hematopoietic cells, most, if not all innate-like CD8+ T cells express high levels of CD44 (CD44high) and CD122 (10, 15). (1). Other distinct features of innate-like T cells is their complete dependence on IL-15 for their development and/or maintenance (16) and the expression of NK cell markers (17). It is noted that in IL-15-deficient mice, there is a dramatic reduction in conventional antigen-experienced memory CD8+ T cells (18-20). IL-15 is required for keeping conventional memory CD8+ T cells in cycle allowing them to respond more rapidly to antigens than naïve cells. It is therefore of great interest to determine if innate-like CD8+ T cells utilize the same mechanism for their survival and rapid induction of effector molecules (21, 22). It is likely that a combination of both the constitutive expression of transcription factors encoding for effector molecules, as well as the slow rate of antigen-independent proliferation observed in MHC class Ib-  157 restricted T cells that endow H2-M3-restricted T cells with the ability to mediate rapid effector functions. These unique properties of H2-M3-restricted T cells make them attractive vaccine targets that can be exploited to influence adaptive immune responses by changing the maturation status of DCs, as demonstrated in this thesis. It is clear from the studies presented in this thesis that some T cell subsets restricted by MHC class Ib molecules, particularly H2-M3 and Qa-1b, share some similarities with antigen-experienced memory CD8+ T cells. Conventional memory CD8+ T cells exist as either effector-memory T cells (CD8EM) or central-memory T cells (CD8CM), the former being more cytolytic and the latter having the ability to stimulate antigen-carrying DCs to augment cell-mediated immune responses. It would be interesting to determine if MHC class Ib restricted T cells are functionally similar to CD8CM T cells (23-25). If CD8CM T cells possess functions that are similar to H2-M3restricted T cells, this would provide an opportunity to target these antigen-experienced CD8CM T cells to augment CD4+ T cell responses via DC vaccination strategies. A potential model system that can be used is based on the varicella-zoster virus (VZV), which causes chickenpox. Immunization with live attenuated varicella vaccine induces a memory T cell response against the immediate early 62 (IE62) protein of VZV that can persist for decades (26-29). Targeting this epitope would result in a more effective vaccination procedure that is linked to more efficient activation of T helper cells, and at the same time ensure that a majority of the human population can benefit from this novel vaccination technique.  158 4.1.2 Significance of a heterogeneous memory CD8+CD44high T cell population Nonconventional CD8+ T cells that can perform a multitude of diverse immune functions have been identified to reside within the memory CD8+CD44high population of T cells. Despite being maintained under germfree conditions with a liquid diet, approximately 10% of CD8+ T cells found in naïve mice displays a memory phenotype, which supports the contention that their development is not due to exposure to environmental antigens (30). As a consequence of this memory phenotype, which is associated with high expression of CD122, these cells can proliferate in an antigen-independent manner in response to stimulation with IL-2 or IL-15 (17). Upon activation by cytokines, these cells express several NK cell receptors, such as NKG2D, CD94, 2B4 and CD16, which can function independently or in concert with the TCR, leading to cytokine production and/or the killing of syngeneic tumor cells (17). Additionally, CD8+CD44high T cells were found to provide early sources of IFN-γ, a proinflammatory cytokine that can amplify innate and adaptive immune responses (31). Interestingly, this early burst of IFN-γ has been attributed to T cells restricted by MHC class Ib molecules. Since IFN-γ can act in synergy with LPS to augment proinflammatory cytokine production (32), induces respiratory burst in phagocytes (33), and aid in the development and shaping of adaptive immune responses as demonstrated in this thesis, it would be of great interest to confirm that at least a significant proportion of CD8+CD44high T cells are in fact restricted by MHC class Ib molecules and that they can perform similar adjuvant-like functions as H2-M3-restricted T cells. Work with B6.Kb-/-Db-/- mice, which are deficient for all MHC class Ia molecules, revealed an even higher percentage (50-60%) of CD8+ T cells that express high levels  159 of CD44 in unmanipulated mice (34). Despite having at least a 90% reduction in the number of mature MHC class Ia-restricted CD8+ T cells in their periphery, B6.Kb-/-Db-/mice contain fully functional CD8+ T cells that can mediate effective immune responses against pathogenic infections (35). For instance, B6.Kb-/-Db-/- mice challenged with LM generated a protective antigen-specific T cell response restricted by the MHC class Ib molecule, H2-M3 (36). In addition to providing protection upon recognizing their cognate antigen, about 80% of CD8+CD44high T cells from B6.Kb-/-Db-/- mice were found to secrete IFN-γ in response to IL-12 and IL-18 since neutralization of these two cytokines greatly reduced the percentages of IFN-γ-producing cells (37). Although the origin and function of these nonconventional CD8+CD44high T cells from normal B6 mice are still largely unknown (10, 38), analysis of the Vβ usage between normal B6 CD8+CD44high T cells and B6.Kb-/-Db-/- CD8+ T cells reveal similar levels of diversity. Furthermore, since B6.Kb-/-Db-/- mice lack MHC class Ia molecules and these mice possess nonconventional CD8+CD44high T cells, these studies strongly suggesting that they are indeed selected by non-MHC class Ia molecules (37). A lack of biased Vβ chain usage by nonconventional CD8+CD44high T cells also suggest that they represent a heterogeneous cell population consisting of T cells restricted by heterogeneous MHC class Ib or other non-MHC class Ia molecules. The ability for rapid IFN-γ production by a heterogeneous cell population, which has the ability to respond with or without TCR ligation to provide both specific and non-specific protection, would be beneficial for the host. Since antigen-specific conventional memory CD8+ T cells represent a relatively small pool of memory cells that are able to mediate bystander activation during some types of pathogenic infections (39-41), it would be of interest to determine if the  160 heterogeneous population of nonconventional cells, consisting of CD8+CD44high T cells, MHC class Ib-restricted T cells, and possibly other subsets, would also provide significant bystander help in immune responses against pathogenic infections. Recent work by two independent groups demonstrated the Tec kinase family members Itk and Rlk were not required for the generation of CD8+ T cells that possess a memory phenotype and innate effector functions (42, 43). Of significant interest, Atherly et al. found that Tec-kinase-deficient CD8+ T cells express high amounts of the transcription factor eomesodermin, which is associated with the ability of memory CD8+ T cells to produce early innate sources of IFN-γ. Interestingly, Broussard et al. reported that Tec-kinase-deficient CD8+ T cells can also be selected by hematopoietic cells, which is similar to the selection of T cells restricted by MHC class Ib molecules, such as H2-M3 and Qa-1b. Future experiments should confirm whether eomesodermin is in fact expressed at higher levels in T cells restricted by MHC class Ib molecules. These experiments should be conducted in either normal B6 or B6.Kb-/-Db-/- mice in order to rule out potential artifacts resulting from Tec kinase deficiency. Once confirmed, it would be of great interest to examine whether this transcription factor is required for the generation of MHC class Ib-restricted T cells, as well as other T cells that possess innate-like properties. Since mice deficient for eomesodermin are embryonic lethal (44), a T cell-conditional knockout would be needed for these studies. Information garnered from these experiments would provide a greater understanding regarding the developmental requirements for the generation of conventional and nonconventional CD8+ T cells.  161 4.2 The in vivo significance of MHC class Ib-restricted T cells  4.2.1 Danger signals mediated by MHC class Ib molecules At the heart of the immune system is the ability to distinguish between self and nonself. The pioneering work of Frank MacFarlane Burnett in the 1940s and 1950s introduced this concept, which he termed the “self-nonself” model, where cells of the immune system responds to foreign antigens, yet are tolerant to antigens derived from self tissues (45). This model was later improved upon by Charles Janeway who in 1989 recognized the importance of antigen presenting cells in the induction of adaptive immune responses, even against self-proteins. He developed the “infectious-nonself” model, which was supported by the discovery of evolutionary conserved membranebound Toll-like receptors (TLRs) that function as pattern-recognition receptors (PRRs) that are able to bind a wide range of pathogen-associated molecular patterns (PAMPs) (46, 47). This concept was slightly modified by Polly Matzinger who introduced the concept that the immune response is not initiated by antigens derived from self or nonself, but rather in response to “danger signals” or endogenous molecules released as a result of damaged tissues (48-50). According to this modified theory, DCs are licensed to initiate an immune response only when they receive signals from injured or stressed tissue cells and not due to cells undergoing apoptosis as a result of normal cellular turnover (49, 50). Unlike healthy steady state conditions, where danger signal-associated molecules would be sequestered, the disruption of normal tissue homeostasis by injury or stress  162 would trigger the release of alarm signals to alert the immune system. These danger signals have been identified as endogenous molecules, which include uric acid, RNA and DNA from apoptotic cells, NKG2D ligands, as well as heat shock proteins (HSPs), (49). Specifically, uric acid is released from injured cells under metabolic stress. Importantly, uric acid has been found to induce DC maturation when co-injected with antigen in vivo and significantly enhances the immune response of conventional CD8+ T cells against tumors, (51, 52). Very recently, eukaryotic RNA and DNA were found to activate DCs via TLR3 and TLR9, respectively, when stressful or necrotic death abruptly exposes these danger signals, which under normal circumstances are confined within cells and not accessible to the immune system (53, 54). The immune system can also be alerted to abnormal tissues that upregulates expression of ligands for NKG2D, which is a receptor on NK cells and a subset of T cells that detect stressed and injured cells (17, 55, 56). Lastly, HSPs, which are over-expressed when tissues are stressed by a number of mechanisms including high temperatures and tumorigenesis, were found to induce DC maturation as well as augment antigen presentation pathways (57-60). Interestingly, T cells restricted by the MHC class Ib molecule, Qa-1, which is the functional counterpart of human HLA-E molecules, were also found to be activated and have the ability to secrete proinflammatory cytokines after recognizing HSPs (61). In this thesis I have shown that H2-M3- and Qa-1-restricted CD8+ T cells possess similar innate-like functions. The recognition of N-formylated peptides presented by H2-M3 molecules may represent a form of endogenously-derived danger signal. It would be interesting to determine whether the normal function of Qa-1b molecules is for the presentation of danger signals in stress or injured cells.  163 Endogenous danger signals have common features that include: i) their ability to become upregulated, released or modified in injured or stressed cells; ii) their confinement to compartments where they can be actively or passively released following injury; iii) their ability to be sensed by a receptor or a DC-related detection system that induced maturation; and iv) their ability to induce effective DC maturation (49). Based on these features, it would be very tempting to speculate that the ability of H2-M3 molecules to present N-formylated peptides may itself be an endogenous danger signal camouflaging as a sensor for bacterial infections given that they initiate protein synthesis with N-formyl-methionine. One such peptide is derived from the Nterminus of NADH dehydrogenase subunit 1 (ND1) in rodents (62). Since the modulation of mitochondrial outer membrane integrity is a key event in the switch between survival and death, the release of a source of mitochondrial N-formylated peptides would be an ideal danger signal since they would only be released when the integrity of the mitochondrial membrane is compromised. In support of this, studies have found that cellular stress induced by oligomycin, an inhibitor of mitochondrial ATPase, which interferes with intracellular calcium mobilization and increases mitochondrial protein release, strongly increased antigen display by H2-M3 molecules (63). Furthermore, to facilitate an immune response by inducing DC maturation, the loss of membrane potential and subsequent release of associated N-formylated peptides must quickly be detected by a receptor or detection system, which in this scenario may be H2-M3 molecules. Consistent with this idea, studies have demonstrated that the majority of H2-M3 molecules are retained in an immature peptide-receptive state held in the ER or early Golgi apparatus and they traffic rapidly to the cell surface upon  164 detecting N-formylated peptides (64). Together with the observation that activated H2M3-restricted T cells express NKG2D, which function as a surveillance receptor for detecting transformed or stressed cells and the work described in this thesis that demonstrates the ability of H2-M3-restricted T cells to induce DC maturation, the hypothesis that H2-M3 molecules may function as a vehicle for presenting endogenous danger signals to the immune system is reasonable. Perhaps the first step to test this hypothesis would be the measurement of stress-induced DC maturation in H2-M3 deficient mice, which have been demonstrated to have compromised innate immune functions (65). 4.2.2 Possible human equivalent of H2-M3 molecules Although humans do not possess a murine H2-M3 counterpart, they do have two homologous N-formyl peptide receptors (FPR) referred to as N-formyl peptide receptorlike 1 (FPRL1) and N-formyl peptide receptor-like 2 (FPRL2) (66). FPRL1 has recently been identified to be a promiscuous receptor that is activated by N-formyl peptides originating from either an endogenous source, such as mitochondrial proteins of ruptured host cells, or an exogenous source such as the proteins of invading pathogens (67, 68). These human FPRs may function similarly to H2-M3 molecules, since they can induce the production of superoxide ions and the release of anti-microbial peptides from phagocytes. Furthermore, FPR-deficient mice exhibit reduced resistance to infection by LM (69). Human FPRs have also been found to interact with structurally diverse proand anti-inflammatory ligands associated with different diseases, including amyloidosis, Alzheimer’s disease, prion disease and HIV (67). It would be of interest to establish whether human FPRs are indeed functional equivalents of murine H2-M3 molecules.  165 This possibility would infer that human FPRs may also be involved in the modulation of downstream adaptive immune responses, which could thus be a property that can be exploited for more effective vaccine designs in humans. In order for this to be achieved, a measurable CD8+ T cell response would first need to be identified. 4.2.3 The autoimmune potential of MHC class Ib-restricted T cells Since T cells restricted by MHC class Ib molecules can recognize endogenous sources of antigens, the validation of MHC class Ib molecules as danger signals would introduce the possibility of tissue-specific autoimmune responses if immune responses to these stress signals are uncontrolled. Studies with Salmonella typhimurium infection have revealed the autoimmune potential of Qa-1b-restricted T cells that recognizes the immunodominant GroEL molecule from this pathogen, which cross-reacted with hostderived HSP60 (61). Furthermore, B6.Kb-/-Db-/- mice that are also deficient for CIITA-/and which lack CD4+ T cells, develop a form of autoimmunity that is characterized by a lymphoproliferative syndrome with notable inflammatory bowel disease and insulitis (70). It is therefore conceivable that MHC class Ib-restricted T cells can also be involved in certain types of autoimmune disease. Although this study is confounded by the absence of CD4+ Treg cells, which in their absence can cause an autoimmunity phenotype even in MHC class Ia-sufficient mice.  4.3 Concluding remarks This thesis demonstrates the ability of T cells restricted by H2-M3- and Qa-1b molecules to function in an adjuvant-like manner to influence the outcome of adaptive T cell  166 immune responses following recognition of N-formylated or stress-induced peptides, respectively. These cells exhibit an activated memory-like phenotype, which together with constitutively expressed transcription factors, may provide them with the ability to respond with innate-like kinetics upon sensing their cognate antigens due to infections or cell stress. Cell contact-dependent molecular interactions and cytokines produced by these activated nonclassical CD8+ T cells induce the maturation of DCs, which in turn expands the numbers and function of both innate and adaptive lymphocytes. As evident by the results presented in this thesis, the interaction of DCs with T cells restricted by MHC class Ib molecules represents a significant control mechanism for immunity that is independent of TLR ligands. The prospect that H2-M3 molecules are vehicles that carry “danger signals” to alert the host to altered self-recognition may enable DC maturation in response to tumors or other stresses, which may not express TLR ligands. Furthermore, unlike the ability of TLR ligands to trigger systemic responses, the maturation of DCs in response to these MHC class Ib-restricted T cells is likely more localized and therefore better at focusing their adjuvant-like characteristics in the context of alerting the host to the presence of infection or cellular stress. Collectively, the results presented in this thesis highlight a novel idea for exploiting the innate-like characteristics of MHC class Ib-restricted CD8+ T cells for generating more effective vaccines against pathogenic infections and possibly cancer, since immune responses against cancer is more dependent on CD4 T cell help. Major goals for the future involve the identification of human T cell subsets that have similar adjuvant-like functions, which can then be harnessed for generating more effective immune responses against pathogenic infections and cancer in humans.  167 4.4 References  1.  Chow, M. T., S. Dhanji, J. Cross, P. Johnson, and H. S. Teh. 2006. H2-M3restricted T cells participate in the priming of antigen-specific CD4+ T cells. J Immunol 177:5098-5104.  2.  Palmer, E. 2003. Negative selection--clearing out the bad apples from the T-cell repertoire. Nature Reviews Immunology 3:383-391.  3.  Medzhitov, R., and C. Janeway, Jr. 2000. Innate immunity. The New England journal of medicine 343:338-344.  4.  Seder, R. A., and R. Ahmed. 2003. Similarities and differences in CD4+ and CD8+ effector and memory T cell generation. Nature immunology 4:835-842.  5.  Rocha, B., and C. Tanchot. 2004. CD8 T cell memory. Seminars in immunology 16:305-314.  6.  Kaech, S. M., E. J. Wherry, and R. Ahmed. 2002. Effector and memory T-cell differentiation: implications for vaccine development. Nature Reviews Immunology 2:251-262.  7.  Das, G., S. Sheridan, and C. A. Janeway, Jr. 2001. The source of early IFNgamma that plays a role in Th1 priming. J Immunol 167:2004-2010.  8.  Bendelac, A., M. N. Rivera, S. H. Park, and J. H. Roark. 1997. Mouse CD1specific NK1 T cells: development, specificity, and function. Annual review of immunology 15:535-562.  9.  Townsend, M. J., A. S. Weinmann, J. L. Matsuda, R. Salomon, P. J. Farnham, C. A. Biron, L. Gapin, and L. H. Glimcher. 2004. T-bet regulates the terminal  168 maturation and homeostasis of NK and Valpha14i NKT cells. Immunity 20:477494. 10.  Urdahl, K. B., J. C. Sun, and M. J. Bevan. 2002. Positive selection of MHC class Ib-restricted CD8(+) T cells on hematopoietic cells. Nature immunology 3:772779.  11.  Sullivan, B. A., P. Kraj, D. A. Weber, L. Ignatowicz, and P. E. Jensen. 2002. Positive selection of a Qa-1-restricted T cell receptor with specificity for insulin. Immunity 17:95-105.  12.  Treiner, E., L. Duban, S. Bahram, M. Radosavljevic, V. Wanner, F. Tilloy, P. Affaticati, S. Gilfillan, and O. Lantz. 2003. Selection of evolutionarily conserved mucosal-associated invariant T cells by MR1. Nature 422:164-169.  13.  Bix, M., M. Coles, and D. Raulet. 1993. Positive selection of V beta 8+ CD4-8thymocytes by class I molecules expressed by hematopoietic cells. The Journal of experimental medicine 178:901-908.  14.  Bendelac, A. 1995. Positive selection of mouse NK1+ T cells by CD1-expressing cortical thymocytes. The Journal of experimental medicine 182:2091-2096.  15.  Dutton, R. W., L. M. Bradley, and S. L. Swain. 1998. T cell memory. Annual review of immunology 16:201-223.  16.  Ohteki, T. 2002. Critical role for IL-15 in innate immunity. Current molecular medicine 2:371-380.  17.  Dhanji, S., and H. S. Teh. 2003. IL-2-activated CD8+CD44high cells express both adaptive and innate immune system receptors and demonstrate specificity for syngeneic tumor cells. J Immunol 171:3442-3450.  169 18.  Lodolce, J. P., D. L. Boone, S. Chai, R. E. Swain, T. Dassopoulos, S. Trettin, and A. Ma. 1998. IL-15 receptor maintains lymphoid homeostasis by supporting lymphocyte homing and proliferation. Immunity 9:669-676.  19.  Khan, I. A., M. Moretto, X. Q. Wei, M. Williams, J. D. Schwartzman, and F. Y. Liew. 2002. Treatment with soluble interleukin-15Ralpha exacerbates intracellular parasitic infection by blocking the development of memory CD8+ T cell response. The Journal of experimental medicine 195:1463-1470.  20.  Kennedy, M. K., M. Glaccum, S. N. Brown, E. A. Butz, J. L. Viney, M. Embers, N. Matsuki, K. Charrier, L. Sedger, C. R. Willis, K. Brasel, P. J. Morrissey, K. Stocking, J. C. Schuh, S. Joyce, and J. J. Peschon. 2000. Reversible defects in natural killer and memory CD8 T cell lineages in interleukin 15-deficient mice. The Journal of experimental medicine 191:771-780.  21.  Gray, D., and P. Matzinger. 1991. T cell memory is short-lived in the absence of antigen. The Journal of experimental medicine 174:969-974.  22.  Zhang, X., S. Sun, I. Hwang, D. F. Tough, and J. Sprent. 1998. Potent and selective stimulation of memory-phenotype CD8+ T cells in vivo by IL-15. Immunity 8:591-599.  23.  Sallusto, F., D. Lenig, R. Forster, M. Lipp, and A. Lanzavecchia. 1999. Two subsets of memory T lymphocytes with distinct homing potentials and effector functions. Nature 401:708-712.  24.  Masopust, D., V. Vezys, A. L. Marzo, and L. Lefrancois. 2001. Preferential localization of effector memory cells in nonlymphoid tissue. Science (New York, N.Y 291:2413-2417.  170 25.  Lanzavecchia, A., and F. Sallusto. 2002. Progressive differentiation and selection of the fittest in the immune response. Nature Reviews Immunology 2:982-987.  26.  Sharp, M., K. Terada, A. Wilson, S. Nader, P. E. Kinchington, W. T. Ruyechan, J. Hay, and A. M. Arvin. 1992. Kinetics and viral protein specificity of the cytotoxic T lymphocyte response in healthy adults immunized with live attenuated varicella vaccine. The Journal of infectious diseases 165:852-858.  27.  Arvin, A. M., E. Kinney-Thomas, K. Shriver, C. Grose, C. M. Koropchak, E. Scranton, A. E. Wittek, and P. S. Diaz. 1986. Immunity to varicella-zoster viral glycoproteins, gp I (gp 90/58) and gp III (gp 118), and to a nonglycosylated protein, p 170. J Immunol 137:1346-1351.  28.  Arvin, A. M., M. Sharp, S. Smith, C. M. Koropchak, P. S. Diaz, P. Kinchington, W. Ruyechan, and J. Hay. 1991. Equivalent recognition of a varicella-zoster virus immediate early protein (IE62) and glycoprotein I by cytotoxic T lymphocytes of either CD4+ or CD8+ phenotype. J Immunol 146:257-264.  29.  Arvin, A. M., M. Sharp, M. Moir, P. R. Kinchington, M. Sadeghi-Zadeh, W. T. Ruyechan, and J. Hay. 2002. Memory cytotoxic T cell responses to viral tegument and regulatory proteins encoded by open reading frames 4, 10, 29, and 62 of varicella-zoster virus. Viral immunology 15:507-516.  30.  Dobber, R., A. Hertogh-Huijbregts, J. Rozing, K. Bottomly, and L. Nagelkerken. 1992. The involvement of the intestinal microflora in the expansion of CD4+ T cells with a naive phenotype in the periphery. Developmental immunology 2:141150.  171 31.  Kambayashi, T., E. Assarsson, A. E. Lukacher, H. G. Ljunggren, and P. E. Jensen. 2003. Memory CD8+ T cells provide an early source of IFN-gamma. J Immunol 170:2399-2408.  32.  Paludan, S. R. 2000. Synergistic action of pro-inflammatory agents: cellular and molecular aspects. Journal of leukocyte biology 67:18-25.  33.  Cassatella, M. A., F. Bazzoni, R. M. Flynn, S. Dusi, G. Trinchieri, and F. Rossi. 1990. Molecular basis of interferon-gamma and lipopolysaccharide enhancement of phagocyte respiratory burst capability. Studies on the gene expression of several NADPH oxidase components. The Journal of biological chemistry 265:20241-20246.  34.  Seaman, M. S., B. Perarnau, K. F. Lindahl, F. A. Lemonnier, and J. Forman. 1999. Response to Listeria monocytogenes in mice lacking MHC class Ia molecules. J Immunol 162:5429-5436.  35.  Vugmeyster, Y., R. Glas, B. Perarnau, F. A. Lemonnier, H. Eisen, and H. Ploegh. 1998. Major histocompatibility complex (MHC) class I KbDb -/- deficient mice possess functional CD8+ T cells and natural killer cells. Proceedings of the National Academy of Sciences of the United States of America 95:12492-12497.  36.  Seaman, M. S., C. R. Wang, and J. Forman. 2000. MHC class Ib-restricted CTL provide protection against primary and secondary Listeria monocytogenes infection. J Immunol 165:5192-5201.  37.  Su, J., R. E. Berg, S. Murray, and J. Forman. 2005. Thymus-dependent memory phenotype CD8 T cells in naive B6.H-2Kb-/-Db-/- animals mediate an antigenspecific response against Listeria monocytogenes. J Immunol 175:6450-6457.  172 38.  Yamada, H., G. Matsuzaki, Q. Chen, Y. Iwamoto, and K. Nomoto. 2001. Reevaluation of the origin of CD44(high) "memory phenotype" CD8 T cells: comparison between memory CD8 T cells and thymus-independent CD8 T cells. European journal of immunology 31:1917-1926.  39.  Zarozinski, C. C., and R. M. Welsh. 1997. Minimal bystander activation of CD8 T cells during the virus-induced polyclonal T cell response. The Journal of experimental medicine 185:1629-1639.  40.  Murali-Krishna, K., J. D. Altman, M. Suresh, D. J. Sourdive, A. J. Zajac, J. D. Miller, J. Slansky, and R. Ahmed. 1998. Counting antigen-specific CD8 T cells: a reevaluation of bystander activation during viral infection. Immunity 8:177-187.  41.  Andreasen, S. O., J. P. Christensen, O. Marker, and A. R. Thomsen. 1999. Virusinduced non-specific signals cause cell cycle progression of primed CD8(+) T cells but do not induce cell differentiation. International immunology 11:14631473.  42.  Atherly, L. O., J. A. Lucas, M. Felices, C. C. Yin, S. L. Reiner, and L. J. Berg. 2006. The Tec family tyrosine kinases Itk and Rlk regulate the development of conventional CD8+ T cells. Immunity 25:79-91.  43.  Broussard, C., C. Fleischacker, R. Horai, M. Chetana, A. M. Venegas, L. L. Sharp, S. M. Hedrick, B. J. Fowlkes, and P. L. Schwartzberg. 2006. Altered development of CD8+ T cell lineages in mice deficient for the Tec kinases Itk and Rlk. Immunity 25:93-104.  44.  Russ, A. P., S. Wattler, W. H. Colledge, S. A. Aparicio, M. B. Carlton, J. J. Pearce, S. C. Barton, M. A. Surani, K. Ryan, M. C. Nehls, V. Wilson, and M. J.  173 Evans. 2000. Eomesodermin is required for mouse trophoblast development and mesoderm formation. Nature 404:95-99. 45.  Burnet, F. M. 1959. Clonal selection theory of acquired immunity. Vanderbilt University Press; Nashville, TN.  46.  Janeway, C. A., Jr. 1992. The immune system evolved to discriminate infectious nonself from noninfectious self. Immunology today 13:11-16.  47.  Janeway, C. A., Jr., and R. Medzhitov. 1998. Introduction: the role of innate immunity in the adaptive immune response. Seminars in immunology 10:349350.  48.  Gallucci, S., and P. Matzinger. 2001. Danger signals: SOS to the immune system. Current opinion in immunology 13:114-119.  49.  Matzinger, P. 2002. The danger model: a renewed sense of self. Science (New York, N.Y 296:301-305.  50.  Matzinger, P. 1994. Tolerance, danger, and the extended family. Annual review of immunology 12:991-1045.  51.  Shi, Y., J. E. Evans, and K. L. Rock. 2003. Molecular identification of a danger signal that alerts the immune system to dying cells. Nature 425:516-521.  52.  Hu, D. E., A. M. Moore, L. L. Thomsen, and K. M. Brindle. 2004. Uric acid promotes tumor immune rejection. Cancer research 64:5059-5062.  53.  Kariko, K., H. Ni, J. Capodici, M. Lamphier, and D. Weissman. 2004. mRNA is an endogenous ligand for Toll-like receptor 3. The Journal of biological chemistry 279:12542-12550.  174 54.  Barrat, F. J., T. Meeker, J. Gregorio, J. H. Chan, S. Uematsu, S. Akira, B. Chang, O. Duramad, and R. L. Coffman. 2005. Nucleic acids of mammalian origin can act as endogenous ligands for Toll-like receptors and may promote systemic lupus erythematosus. The Journal of experimental medicine 202:1131-1139.  55.  Gasser, S., and D. H. Raulet. 2006. The DNA damage response arouses the immune system. Cancer research 66:3959-3962.  56.  Raulet, D. H. 2003. Roles of the NKG2D immunoreceptor and its ligands. Nature Reviews Immunology 3:781-790.  57.  Callahan, M. K., M. Garg, and P. K. Srivastava. 2008. Heat-shock protein 90 associates with N-terminal extended peptides and is required for direct and indirect antigen presentation. Proceedings of the National Academy of Sciences of the United States of America 105:1662-1667.  58.  Liberek, K., A. Lewandowska, and S. Zietkiewicz. 2008. Chaperones in control of protein disaggregation. The EMBO journal 27:328-335.  59.  Vanaja, D. K., M. E. Grossmann, E. Celis, and C. Y. Young. 2000. Tumor prevention and antitumor immunity with heat shock protein 70 induced by 15deoxy-delta12,14-prostaglandin J2 in transgenic adenocarcinoma of mouse prostate cells. Cancer research 60:4714-4718.  60.  Javid, B., P. A. MacAry, and P. J. Lehner. 2007. Structure and function: heat shock proteins and adaptive immunity. J Immunol 179:2035-2040.  61.  Lo, W. F., A. S. Woods, A. DeCloux, R. J. Cotter, E. S. Metcalf, and M. J. Soloski. 2000. Molecular mimicry mediated by MHC class Ib molecules after infection with gram-negative pathogens. Nature medicine 6:215-218.  175 62.  Wang, C. R., A. R. Castano, P. A. Peterson, C. Slaughter, K. F. Lindahl, and J. Deisenhofer. 1995. Nonclassical binding of formylated peptide in crystal structure of the MHC class Ib molecule H2-M3. Cell 82:655-664.  63.  Hermel, E., E. Grigorenko, and C. J. Aldrich. 1997. Increased class Ib antigen display on TAP-2 mutant cells by a mitochondrial function inhibitor. Cellular immunology 179:10-15.  64.  Princiotta, M. F., L. L. Lenz, M. J. Bevan, and U. D. Staerz. 1998. H2-M3 restricted presentation of a Listeria-derived leader peptide. The Journal of experimental medicine 187:1711-1719.  65.  Xu, H., T. Chun, H. J. Choi, B. Wang, and C. R. Wang. 2006. Impaired response to Listeria in H2-M3-deficient mice reveals a nonredundant role of MHC class Ibspecific T cells in host defense. The Journal of experimental medicine 203:449459.  66.  Murphy, P. M., T. Ozcelik, R. T. Kenney, H. L. Tiffany, D. McDermott, and U. Francke. 1992. A structural homologue of the N-formyl peptide receptor. Characterization and chromosome mapping of a peptide chemoattractant receptor family. The Journal of biological chemistry 267:7637-7643.  67.  Le, Y., P. M. Murphy, and J. M. Wang. 2002. Formyl-peptide receptors revisited. Trends in immunology 23:541-548.  68.  Ying, G., P. Iribarren, Y. Zhou, W. Gong, N. Zhang, Z. X. Yu, Y. Le, Y. Cui, and J. M. Wang. 2004. Humanin, a newly identified neuroprotective factor, uses the G protein-coupled formylpeptide receptor-like-1 as a functional receptor. J Immunol 172:7078-7085.  176 69.  Gao, J. L., E. J. Lee, and P. M. Murphy. 1999. Impaired antibacterial host defense in mice lacking the N-formylpeptide receptor. The Journal of experimental medicine 189:657-662.  70.  Das, G., J. Das, P. Eynott, Y. Zhang, A. L. Bothwell, L. Van Kaer, and Y. Shi. 2006. Pivotal roles of CD8+ T cells restricted by MHC class I-like molecules in autoimmune diseases. The Journal of experimental medicine 203:2603-2611.  177 Appendix A: List of publications  I share co-authorship on four other papers that are not included in this thesis: Dhanji, S., Chow, M.T., and Teh, H.S. Self-antigen maintains the innate antibacterial function of self-specific CD8 T cells in vivo. J. Immunol. 2006. 177: 138-146. Chen, X., Priatel, J.J., Chow, M.T., and Teh, H.S. Preferential development of CD4 and CD8 T regulatory cells in RasGRP1-deficient mice. J. Immunol. 2008. 180:5973-5982. Kim, E.Y., Teh, S.J., Yang, J., Chow, M.T., and Teh, H.S. J. TNFR2-deficient memory CD8 T cells provide superior protection against tumor cell growth. J. Immunol. 2009. In Press. Priatel, J.J., Chen, X., Huang, Y.H., Chow, M.T., Zenewicz, L.A., Coughlin, J.J., Shen, H., Stone, J.C., Tan, R., and Teh, H.S. RasGRP1 regulates antigen-induced developmental programming by naïve CD8 T cells. Resubmitted following revisions.  For these four papers I contributed 10% to the experimental design, 10% of the research and 10% of the data analysis.  178 Appendix B: UBC research certificates of approval  179  

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